http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Achim&year=&month=2010.igem.org - User contributions [en]2024-03-28T12:56:42ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Freiburg_Bioware/SafetyTeam:Freiburg Bioware/Safety2010-10-28T03:22:20Z<p>Achim: </p>
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<h1 style="text-align: justify;">Biosafety</h1><br />
<p style="text-align: justify;">Definition: "biosafety measures aim to<br />
prevent the unintentional<br />
exposure to pathogens and toxins, or their accidental release" <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br />
</p><br />
<h2 style="text-align: justify;">Risk assessment for our<br />
Adeno-associated virus based system</h2><br />
<p style="text-align: justify;">In Germany the "Central Commission for<br />
Biological Security" (ZKBS) released three legally binding Risk<br />
Assessment satements for the Adeno-associated Virus <sup>26,</sup><br />
<sup>27,</sup><br />
<sup>28</sup>.<br />
Risk assessment in other countries may deviate from these decisions, so<br />
please inform yourself about the legal regulations on AAV<br />
in your country before using the Virus Construction Kit.<br><br />
</p><br />
<div style="text-align: justify;">In Germany the Biological Safety<br />
Level(BSL) classifications for AAVs are:<br />
</div><br />
<ul style="text-align: justify;"><br />
<li>Adeno-associated Virus 2, 3 and 5 have to be handled under BSL 1.</li><br />
<li>Adeno-associated Viurs 1, 4, 5, 7, 8, 9, 10 and 11 have to be<br />
handled under BSL 2.</li><br />
</ul><br />
<p style="text-align: justify;">This classification was developed based<br />
on the fact that only serotypes 2, 3 and 5 are isolated from humans and<br />
that their harmlessness has been confirmed in clinical studies. This<br />
general classification has to be rechecked when the virus contains gene<br />
sequences with a transforming protential.<br />
</p><br />
<p style="text-align: justify;"><b>Viral vectors systems</b> packaging<br />
a vector plasmid that only contains the<br />
viral Inverted Terminal Repeats (ITRs) and providing the genes for<br />
Rep and Cap in trans (as it is the case for our system) are classified<br />
as <b>BSL 1</b> if the following conditions are fullfilled: </p><br />
<ul style="text-align: justify;"><br />
<li>The viral particles do not contain AAV derived sequences other<br />
than the ITRs</li><br />
<li>The viral particles do not contain Nucleotidesequences with a<br />
risk potential</li><br />
</ul><br />
<p style="text-align: justify;">We also investigated the legal<br />
regulations for AAV-2 Viral Vector<br />
systems in the United States. The guidelines of the National Institutes<br />
of Health (NIH) classify in the <a<br />
href="http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm">Appendix<br />
B</a><sup>32</sup> AAV-2 Vector Systems as Risk Group 1 (RG1) agents<br />
that can be treated under BSL 1. In detail the appendix states "...<br />
adeno- associated virus (AAV) types 1 through 4; and recombinant AAV<br />
constructs, in which the transgene does not encode either a potentially<br />
tumorigenic gene product or a toxin molecule and are produced in the<br />
absence of a helper virus."<br />
</p><br />
<div style="text-align: justify;">Other general observations with the<br />
Adeno-associated Virus 2 AAV-2)are: </div><br />
<ul style="text-align: justify;"><br />
<li>In clinical studies the viral vectors were not dilivered to the<br />
gonades. </li><br />
<li>Vector sequences were not detectable in the patients blood or<br />
urine at an examination 48 h after an infection with AAV-2 <sup>29</sup>.<br />
</li><br />
<li>In absence of Rep proteins, the vector DNA stays extrachromosomal<br />
and is not frequently integrated.</li><br />
</ul><br />
<p style="text-align: justify;"> Concluding all these informations and<br />
regulations, the project that we have designed in this year is clearly<br />
classified as BLS 1.<br />
</p><br />
<div style="text-align: justify;"> </div><br />
<h2 style="text-align: justify;">General biosafety regulation in Germany</h2><br />
<table<br />
style="text-align: left; width: 90%; margin-left: 0px; margin-right: 0px;"<br />
border="0" cellpadding="2" cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><br />
<p><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></p><br />
</td><br />
<td style="vertical-align: middle;"><span<br />
style="color: darkgreen;"><b>Which specific biosafety rules or<br />
guidelines do you have to consider in your country?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div style="text-align: justify;"><br />
</div><br />
<div<br />
style="margin: 0px 5px; width: 700px; float: right; height: auto; text-align: justify;"><span<br />
style="color: darkgreen;"> </span></div><br />
<div style="text-align: justify;"><br />
</div><br />
<p style="text-align: justify;">In Germany all work that includes<br />
recombinant DNA technologies is<br />
regulated by the <a href="http://bundesrecht.juris.de/gentg/index.html">Gesetz<br />
zur Regelung der Gentechnik</a>. This law regulates general<br />
aspects in the life sciences and refers for more precise<br />
interpretations in §4 to the <a<br />
href="http://bundesrecht.juris.de/gentg/__4.html">Zentrale<br />
Kommission für die Biologische Sicherheit</a>. The ZKBS is a<br />
commission composed of 20 technical experts that releases yearly<br />
statements to actual issues of biosafety. So far the ZKBS released<br />
three stratements affecting the work with Adeno-associated viral<br />
systems<br />
<a<br />
href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf"><sup>26,</sup></a><br />
<a<br />
href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf"><sup>27,</sup></a><br />
<a<br />
href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf"><sup>28</sup></a>.<br />
These documents were used to assess the dangers that could arise from<br />
our project to team members and the enviroment.<br />
</p><br />
<table<br />
style="text-align: left; width: 90%; margin-left: 0px; margin-right: 0px;"<br />
border="0" cellpadding="2" cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; width: 209px;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle; width: 778px;"><span<br />
style="color: DarkGreen;"><b>Is there a local biosafety group,<br />
committee, or review board at your institution? If yes, what does your<br />
local biosafety group think about your project?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><span<br />
style="color: DarkGreen;"></span></div><br />
<p style="text-align: justify;">At the Albert-Ludwigs-University<br />
Freiburg for all concerns of security the <a<br />
href="http://www.sicherheit.uni-freiburg.de">Stabsstelle Sicherheit</a><br />
is responsible and to contact if questions arise. Especially for<br />
questions of biological security Dr. Petra Markmeyer-Pieles is<br />
cognizant. We contacted her a first time befor the begin of our project<br />
in March when it was clear that the Adeno-associated Virus (AAV-2) was<br />
chosen as the topic of our project. At that time she proposed to do the<br />
cloning in the AAV-2 that is for sure to handle under biological<br />
security level 1 and to prepare everything for work under biological<br />
security level 2 to satisfy the precaution principle.<br />
The precaution principle was realized and all viral vectors that<br />
contained a modified capsid were handled under SII conditions until<br />
proven harmless.<br />
In August the planing of the project was completed, summarized in an <a<br />
href="https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf">Biosafety<br />
application<sup>30</sup></a> and handed to the department for<br />
biological security who approve the application in an <a<br />
href="https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg">official<br />
BSL1 confirmation<sup>31</sup></a>official BSL1 confirmation for our<br />
project.</p><br />
<h2>Risk management</h2><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: justify;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle; text-align: justify;"><span<br />
style="color: DarkGreen;"><b>Would any of your project ideas raise<br />
safety issues in terms of: researcher safety, public safety, or<br />
environmental safety?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p style="text-align: justify;"><br />
Our project was designed in a way that it avoids any serious safety<br />
issues as far as possible.<br />
When working with infectious particles a minimal risk for the<br />
researcher is allways present. This risk was minimized by restricting<br />
the transduced genes to fluorescent proteins and prodrug convertases<br />
that are already proven not to harm human cells in the absece of the<br />
corresponding prodrug. A potential danger for the public or the<br />
environment was minimized as much as possible by following strictly the<br />
rules of Good Laboratory Practice (GLP) and the abdication of using<br />
randomized insertions in the capsid and of replication potent viruses.<br />
Minimizing the risk for team members and the society was was allways<br />
one of the major concerns, especially because worries about<br />
undergraduate students manipulating a virus could arise.<br />
The security concept will be explained by quoting and explaining the<br />
six guiding principles for safe manipulation of Gene Manipulated<br />
Organisms (GMOs) as summarized in Kimman et al. ; 2008<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/18625678"><sup>18</sup></a>.<br />
</p><br />
<b>1) Hazard recognition and<br />
identification</b><br />
Risk assessment has been done and all legal regulations were considered<br />
as described in the last paragraph.<br />
<b><br><br />
2) Biological containment</b><br />
Biological containment means the usage of organisms with "reduced<br />
replicative capacity, inefectivity , transmissibility, and<br />
virulence"18.<br />
For our project only replicative deficient viruses were used,<br />
additional all modifications aimed to have a specific targeting of the<br />
resulting viral vectors for a specific cell type. This gain in<br />
specificity requires it to cut off the braod natural tropism resulting<br />
is an less infective virus copared to the wild type virus.<br />
<br><br />
<b>3) Concentration and enclosure</b><br />
All working steps for the preparation of viral vectors were carried out<br />
in a BSL II laboratory and within this laboratory work with the AAV was<br />
restricted to a separate Laminar flow cabinet type II. Cell culture and<br />
storage of the virus was also done in separate Freezers and incubators.<br />
All laboratories and epipment that contained viral vectors were<br />
specially marked, for example with a Biohazard warning signs.<br />
<b><br><br />
4) Exposure minimization</b><br />
This aspect of the guiding principles can be sumarized under "operator<br />
protection"18. The Exposure minimization was achieved in our laboratory<br />
by wearing special labcoats for the SII laboratory and gloves that were<br />
desinfected and changed regualrely. During manipulation of viral<br />
vectors attention was payed to avoid droplets and especially aerosoles.<br />
A possible diversion of the viral vectors was avoided by cleaning all<br />
equipment when inserted or removed from the Laminar flow cabinet and<br />
after completion of the work task.<br />
<b><br><br />
5) Physical containment</b><br />
The requirements for the physical containment were fullfilled by<br />
performing all manipulation on the AAV in an BSL II laboratory that<br />
guaranted a restriction of persons that entered the laboratory.<br />
<b><br><br />
6) Hazard minimization</b><br />
For the AAV-2 there are no sugestive activitis because the possible<br />
danger that runs out of the AAV is comparably low, vaccination is not<br />
avilible and biomonitoring is not necessary.<br />
<br><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Do any of the new BioBrick parts (or<br />
devices) that you made this year raise any safety issues? If yes, did<br />
you document these issues in the Registry? How did you manage to handle<br />
the safety issue? How could other teams learn from your experience?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p><br />
</p><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: justify;">Several<br />
composite parts that were assembled by our Team<br />
this year are alone capable of producing infectious viral particles<br />
when transduced together with a vector plasmid and a helper plasmid<br />
into <a<br />
href="https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf">AAV-293</a><br />
cells. These special cells provide the adenoviral gene E1 stabily<br />
integrated in trans.<br />
These cells are not provided in the Virus Construction Kit nor<br />
availible in the Parts Registry and have to purchased from other<br />
laboratories or a commercial supplyer.<br />
For this<br />
reason we estimate the risk of a accidental transformation of <a<br />
href="https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf">AAV-293</a><br />
cells with all three plasmids for negligible.<br />
Nevertheless we considered it useful to mark every BioBrick or<br />
Composite Part in the Registry that contributes to the production or is<br />
capable of producing viral vectors when transformed under the<br />
previously mentioned conditions.</td><br />
<td style="vertical-align: top;"><br />
<p><img margin:="" 0px="" 5px=""<br />
src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_Warning_SignI.png"<br />
alt="Warning sign for part descriptions" align="right" width="300"></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
</p><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Contribute to community discussions<br />
on what needs to go into a code against the use of our science for<br />
hostile purposes (see A Community Response)</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p><br />
</p><br />
<ul><br />
<li>Public Perception </li><br />
<li>importance of maintaining public legitimacy and support</li><br />
<li>apllications should demonstrate clear social benefits</li><br />
<li>not overhyped - anxiety and unrealistic hopes</li><br />
</ul><br />
<blockquote>psychological research into the concept of "identity-driven<br />
decision-making" (Torpman,2004)<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/16819452"> <sup>19</sup></a></blockquote><br />
<blockquote>Every grout has a set of norms: a code of conduct about<br />
what is acceptable beahviour (Jaques, 2004]<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/16819452"> <sup>19</sup></a></blockquote><br />
<br><br />
<br><br />
<center><br />
<h2>Trade-off between potential misuse and promising medical progress</h2><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: left;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Do you have any other ideas how to<br />
deal with safety issues that could be useful for future iGEM<br />
competitions? How could parts, devices and systems be made even safer<br />
through biosafety engineering?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
</center><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"> </span></div><br />
<p><br />
In principle each research-project that bears any risks for engaged<br />
researchers, mankind or the environment should be treated under the<br />
precautionary principle as proposed <a<br />
href="http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf"><sup>11</sup></a>:<br />
"treat synthetic microorganisms as dangerous until proven harmless".<br><br />
This would mean to work on such synthetic DNA containing Bio Bricks at<br />
least under Biological security levels two.<br><br />
Additional to this secure working environment the system itself can be<br />
optimized according to biosafety aspects, means to reduce it's<br />
viability outside the laboratory. This aim can be approached by<br />
reducing the systems ability to evolve, proliferate and interact with<br />
it's environment. A common method to achieve this goal is to engineer<br />
microorganisms in a way that they depend on nutrients that can't be<br />
found in the environment in sufficient amount.<br />
</p><br />
<br><br />
<br><br />
<center><br />
<h1>Biosecurity</h1><br />
</center><br />
Def: "measures focus on the prevention of theft, misuse , or<br />
intentional relese of pathogens and toxins" <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br><br />
<br><br />
<center><br />
<h2>The malignant use of biological agents in history</h2><br />
</center><br />
<p>The misuse of biological agent as weapons in warfare is a fear<br />
spreading companion in the history of mankind, ranging from the<br />
well-poisoners in prehistoric times to bio-terrorists present days. The<br />
following brakt intends to give a short outline of the major events<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a>:<br />
</p><br />
<ul><br />
<li> First systhematic use of chemical weapons during the first world<br />
war</li><br />
<li> The 1918 flu pandemic lasted from March 1918 to June 1920 and<br />
killed approximately 50 Million people around the world. Rumors<br />
circulated that this pandemic is caused by the other combatant nation.</li><br />
<li> "Prohibion of the Use of Asphyxiating, Poisonous or other Gases<br />
and of Bacteriological Methods of Warfare" was signed on 19 June 1925<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a></li><br />
<li> Several combatant nations of the second world war established<br />
biological warfare programs. </li><br />
<li> The range of the Japanese biological warfare program lead<br />
several<br />
nations to expand their own biological warfare program.</li><br />
<li> Limited military use<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a><br />
and the advances in molecular biology (e.g. the first isolatin of a<br />
gene and the discovery of the restriction enzymes in 1969) lead to the<br />
"Convention on the Prohibion of the Development, Production and<br />
Stockpilling of Bacteriological (Biological) and Toxin Weapons and on<br />
Their Destruction " (BTWC) on 10 April 1972.<br />
<blockquote>''"develop, produce, stockpile or otherwise acquire or<br />
retain: ... Microbial or other biological agents, or toxins whatever<br />
their origin or method of production, of types and in wantities that<br />
have no justification for prophylactic, protective or other peaceful<br />
purpose..." ''<sup>3</sup></blockquote><br />
</li><br />
<li> Breach of the BTWC by the Soviet Union which continued their<br />
offensive biologicla warfare programm </li><br />
<li> Stop of the biological warfare Program "Biopreparat" accompanies<br />
the Dissolution of the Svoviet Union in 1991</li><br />
<li> Antrax attacks in the USA in 2001</li><br />
After this short description of misused biological components there is<br />
still the question what impact biological warfare will have in the<br />
future of mankind.<br><br />
<br><br />
</ul><br />
<center><br />
<h2>Broad avilibility of knowledge</h2><br />
</center><br />
n the life sciences information has allways been freely acessible for<br />
everybody who is interested in the results of a particular research<br />
project. The combination of this global availibility with the new<br />
possibilities of the internet broad for literature search and<br />
availibility of gene sequences made it easiere to collect informations<br />
for a possible misuse. <br><br />
For the overwhelming majority this open availibility is absolutely<br />
desirely but on the other hand there are also examples of research<br />
results that bear a very high risk to be misused. <br><br />
Following we present the three most controversial discussed<br />
publications that could also be read as a "How to create your own<br />
bioweapon".<br />
<h3>Mousepox Virus in Australien</h3><br />
<br><br />
The Australian research group around <a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/11152493"> Jackson et al. ;<br />
2001 <sup>16</sup></a>inserted the coding region of the IL-4 gne into<br />
the genome of mousepox. They hoped to create a virus that sterilizes<br />
mice and thus provides a means for pest control. Contrarely to their<br />
expectations they had created a superstrain that killed even naturally<br />
resistent mice and mice that had been vaccinated against normal<br />
mousepox. This discovery could potentially be used to make smallpox<br />
resistant to potential vaccines.<br />
<h3>Synthesis of a Polio Virus</h3><br />
<br><br />
At the State University of New York <a<br />
href="http://www.sciencemag.org/cgi/content/full/297/5583/1016"> Cello<br />
et al. ; 2002 <sup>05</sup> synthesized a "living" polio virus from<br />
scratch. Especially the </a><a<br />
href="http://www.sciencemag.org/cgi/content/full/1072266/DC1">Supporting<br />
Online Material</a> caused worried remarks because it precisely<br />
describes how to assemble a virus from small oligo nucleotides. The<br />
authors jusifyed their project by declaring that they:<br />
<blockquote>"made the virus to send a warning that terrorists might be<br />
able to make biological weapons without obtaining a natrual virus"<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/19784453"><sup>17</sup></a></blockquote><br />
<h3>Reconstitution of the Spanish Flu</h3><br />
<br><br />
<img style="width: 300px; height: 223px;" class="right"<br />
src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Spanishflu.jpg"<br />
alt="Influenza hospital for american soldiers" align="left"><br />
At the Center for Disease Control and Prevention (CDC) the group around<br />
<a href="http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf">Tumpey<br />
et al. ; 2005<sup>24</sup></a> published that they had sequenced and<br />
recreated the pandemic Spanish Flu Virus of 1918 which killed 20-50<br />
million people. The publication of the sequence provoked several very<br />
different responses that can only be partially be quoted here.<br />
The controversial noticed outrider of the Synthetic Biology Craig<br />
Venter seen in the new virus<br />
<blockquote>"the first true Juressic Parc scenario" <a<br />
href="http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&amp;sID=4233"><sup>2</sup></a></blockquote><br />
The Institute Professor at the MIT Philip A. Sharp supported the<br />
publication because he:<br />
<blockquote>"belive[s] that allowing the publication of this<br />
information was the correct decision in terms of both national security<br />
and public health" <a<br />
href="http://www.sciencemag.org/cgi/content/short/310/5745/17"><sup>12</sup></a></blockquote><br />
Wheras <a<br />
href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf">v.<br />
Bubnoff; 2005<sup>25</sup></a> critisizes the CDS for it's careless<br />
regulations for the shipment of viruses and the willingness to<br />
propagate highly virulent viruses. This causes in his opinion the risk<br />
of possible accidents resulting in the release of the virus to the<br />
environment.<br><br />
<br><br />
<h3>Availability of molecular biological techniques</h3><br />
<br />
The knowledge required for the creation of a genetically engineered virus can easily be accessed from all over the world in online gene banks and publication databases and could be potentially be used for a destructive purpose <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819443"><sup>20</sup></a>. In 2007, Scott C. Mohr published the first part of his manuscript <a href="http://openwetware.org/images/3/3d/SB_Primer_100707.pdf">Primer for Synthetic Biology<sup>14</sup></a></li>. This open accessible document explaining the basics of molecular engineering addresses people interested in genetics but coming from a non-academic background. This development called "garage biology" or "biohacking" is a clear indication for the tendency to have knowledge about molecular biology available in society, as it was seen with programming and computer hacking from ~1980 on.<br />
<br />
<h3>Availability of synthesized DNA</h3><br />
<br />
<br />
One of the reactions to the publication of the genome sequence of the Spanish flu strain from 1918 was <a href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf"> v. Bubnoff, 2005<sup>25</sup></a></li> who pointed out the lax handling of the reconstituted virus sequence, the ease of availability and the low effort that has to be invested to recreate a virus:<br />
<blockquote>''"Scientists in Canada are planning to work with the virus, although they will not request it from the CDC. [...] Reconstructing the live virus from its DNA would then take just a few days, he says.''</blockquote><br />
Technical advances and dropping prices in the field of gene synthesis brings several deadly germs into the range of small research projects or even private persons as for example Spanish Flu (Influenza H<sub>1</sub>N<sub>1</sub>) with a 13.5 kbp <a href=http://expasy.org/viralzone/all_by_species/131.html><sup>*</sup></a> or even the Ebola virus with a 19 kbp genome <a href=http://expasy.org/viralzone/all_by_species/207.html><sup>*</sup></a>.<br />
<br />
In 2006, the “Guardian” journalist James Randson ordered a 78 bp DNA oligonucleotide coding for the capsid of the smallpox <i>Orthopoxvirus variola</i> with the intention to alert the public.<br />
<a href="http://www.guardian.co.uk/world/2006/jun/14/terrorism.topstories3"><sup>10</sup></a> <br><br />
In the United States, smallpox are listed as schedule 5 pathogens in the <i>Anti-terrorism, Crime and Security Act of 2001</i> and are illegal to keep or use without first notifying the civil authorities.<br><br />
On the other hand, it is questionable if these prohibitions or perhaps the size of the smallpox genome of ~200 kb <a href=http://expasy.org/viralzone/all_by_species/149.html><sup>*</sup></a> are sufficient to prevent de novo synthesis in the future efficiently. His intention was to focus the public's attention to the possibility that one of the most severe plagues in the history of mankind could be synthesized and released to the environment, either intentionally or by mistake. This would be a tragic setback since the smallpox's eradication was celebrated in 1979 by the World Health Organization as one of the greatest victories in the history of medicine <a href=http://www.who.int/mediacentre/factsheets/smallpox/en><sup>33</sup></a>.<br><br />
In order to prevent the unauthorized synthesis of gene sequences encoding hazardous biological agents, efforts to implement sequence analysis algorithms into the operating procedure of all organizations and companies capable of synthesizing gene sequences are being undertaken.<br> <br><br />
This barrier should be implemented not on a voluntary basis but as a legally binding regulation. It should be enforced by the government on a national level, but effort should also be made to find solutions for an international progress on this issue.<br><br />
As a first step, the development of search algorithms as Craic's BlackWatch<a<br />
href="https://biotech.craic.com/blackwatch/introduction.html><sup>34</sup></a> should be promoted and refined.<br><br><br />
Additional to this sequence base search for possible misuse of gene sequences, each order of already existing or synthesized genes could first be aligned with a list of countries, and in a second search with the so called Hadex exclusion list that names people and organizations excluded from obtaining dual-use gene material<a href=http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf><sup>1</sup></a>. <br />
<br><br />
<h2>The nature of biological weapons - Who is willing to accept their drawbacks</h2><br />
When considering the use of biological means for warfare, a potential aggressor has to accept several serious drawbacks. Biological weapons are not fully controllable, harmful effects to the civilian population cannot be foreseen. If replication-potent germs were used for such a purpose, they could possibly mutate and seriously harm mankind or other animal populations.<br><br />
This makes biological warfare unattractive for most nations, especially because more predictable weapons exist that can easily be directed against military targets with limited collateral damage.<br />
Unfortunately, national warfare programs are not the only source of danger when considering biological warfare. <br />
<blockquote>"An increasing number of countries believe that their political and security interests could be protected or achieved only through the possession of such weapons, especially in view of the overwhelming superiority of the US armed forces in terms of conventional weapons."<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a></blockquote> <br />
Several countries around the globe are technically capable to develop biological weapons or to support terroristic groups in doing so. When this physical capability meets an ideological attitude that is based on contempt for other concepts of society, it cannot be ruled out that biological warfare is taken into consideration. <br><br><br />
<br />
<h2>Synthetic Biology vs. Nuclear Research - Consideration in terms of security policy<h3> <br />
When considering the history of research, the dual-use character of technical innovations has always been present, only the reaction of society to it differs from case to case. As an example, scientific discoveries in the field of nuclear physics where treated as classified information having a high relevance for the national security as shown in the case of the Manhattan project<a href="http://www.ncbi.nlm.nih.gov/pubmed/19784453"><sup>17</sup></a>. A comparable censorship is not established in life sciences research apart of some cases when national security was endangered.<br><br><br />
Another point concerning the threat arising from biological weapons is the absence of methods that can be used to monitor such weapons of mass destruction as pointed out by M.R. Dando. This fact and the increasing world-wide mobility would make it impossible to prevent the spreading of such weapons when once accessible to potential assassins. <br />
<br />
<h3>Is a revision of basic research necessary in sensitive research fields? </h3><br />
The publication of research papers containing dual-use knowledge caused several people to call for regulation. In the United States, the so called Fink Committee evaluated the possibility that research in life sciences could be used for biological warfare purposes and how this could be avoided.<a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br><br><br />
<br />
This committee had two recommendations:<br />
<ul><br />
<li>To familiarize the scientific community with the dual-use problem in the life sciences.</li><br />
<li>To review experiments of concern. Experiments are of concern if they:<br />
<ul><br />
<li>render a vaccine ineffective</li><br />
<li>confer resistance to therapeutically useful antibiotics or<br />
antiviral agents</li><br />
<li>enhance the virulence of a pathogen or render a nonpathogen<br />
virulent</li><br />
<li>increase transmissibility of a pathogen</li><br />
<li>alter the host range of a pathogen</li><br />
<li>enable the evasion of diagnostic/detection tools</li><br />
<li>enable the weaponization of a biological agent or toxin</li></ul><br />
<li>To review publications with a strong dual-use factor</li><br />
<li>To create a National Science Advisory Board</li><br />
<li>To improve the oversight and reduce unauthorized accessibility of hazardous gene material</li><br />
<li>To include the life sciences into the efforts for national security</li><br />
<li>To harmonize the international oversight over dual-use research</li><br />
</ul><br />
<br />
The implementation of these recommendations would be desirable, even though they might cause inconveniences for scientists working in the affected fields.<br />
<br />
<br />
<center><br />
<h2>Conclusion</h2><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Include<br />
something in your project<br />
description and presentations that demonstrates that you have thought<br />
about how others could misuse your work.</b></span> </td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
</center><br />
<p>For sure there is allway the possibility that knowledge to produce<br />
transgene viral vectors could be used to produce bioweapons. Therefor<br />
it was important for us to use a system that does not bear the risk<br />
that someone could use it for evil purpose. In the case of the<br />
Adeno-associated virus the very limited packaging capacity is the major<br />
reason that excludes it from the list of agents that could<br />
realistically be used for the pruduction of bioweapons. Even a fully<br />
replication potent AAV will depend on the coninfection of a helpervirus<br />
and is therefore not suitable for a fast propagation in an population.<br />
Additional to this point we concentrated our project on the retargeting<br />
of the virus - means to make the broad tropismn more narrow and to<br />
decrease the transduction efficiency in the most cases. This<br />
modification is usually mainly required for medical purposes. Also we<br />
did neither investigate possibilities to shield the vector from the<br />
immune system of potential host nor ways to bypass an existing<br />
immunity.<br><br><br />
<br />
Concluding all factors mentioned above, we need to consider the possibility that a person, organization or state could misuse the fast-advancing life sciences for biological warfare. The possibilities and the simplicity of dual-use research misuse will become more and easier the faster the scientific progress advances.<br><br />
Therefore it is important to minimize these potential risks before they become reality. This is not only the task of a designated group but a moral obligation for scientists, politicians and everybody related to dual-use research. Especially scientists have to contribute to and lead the continuing discussion on this topic because they are able to estimate how aspects of their research fields might be misused.<br />
</p><br />
<br><br />
<br><br />
<ul><br />
<li>01 <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf">Synthetic<br />
Biology &amp; Biosecurity - Awareness in Europe | Kelle ; 2007<sup>1</sup></a></li><br />
<li>02 <a<br />
href="http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&amp;sID=4233">Synthetic<br />
Biology - Social and Ethical Challenges | Balmer et Martin ; 2008<sup>2</sup></a></li><br />
<li>03 Synthetic Biology - Applying Engineering to Biology <br><br />
used </li><br />
<li>04 <a<br />
href="http://www.informaworld.com/smpp/content%7Econtent=a713604665&amp;db=all">The<br />
Impact of the Development of Modern Biology and Medicine on the<br />
Evolution of Offensive Biological Warfare Programs in the Twentieth<br />
Century | Dando ; 1999 <sup>04</sup></a></li><br />
<li>05 <a<br />
href="http://www.sciencemag.org/cgi/content/full/297/5583/1016">Chemical<br />
Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the<br />
Absence of Natural Template | Cello et al. ; 2002 <sup>05</sup></a></li><br />
<li><a href="http://www.sciencemag.org/cgi/content/full/297/5583/1016">06<br />
</a><a<br />
href="http://www.nytimes.com/2003/12/03/opinion/03iht-edstein_ed3_.html">Dangerous<br />
research : When science breeds nightmares | Steinbruner et Harris ;<br />
2003 <sup>06</sup></a></li><br />
<li>07 <a<br />
href="http://www.jcvi.org/cms/fileadmin/site/research/projects/synthetic-genomics-report/synthetic-genomics-report.pdf">Synthetic<br />
Genomics - Options for governance | Garfinkel et al. ; 2007 <sup>07</sup></a></li><br />
<li>08 <a<br />
href="http://www.nature.com/nrg/journal/v6/n7/abs/nrg1637.html">Synthetic<br />
biology | Benner et Sismour , 2005 <sup>08</sup></a></li><br />
<li>09 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16954140">Synthetic<br />
biology—putting engineering into biology | Heinemann et Panke ; 2006 <sup>09</sup></a></li><br />
<li>10 <a<br />
href="http://www.guardian.co.uk/world/2006/jun/14/terrorism.topstories3">Revealed:<br />
the lax laws that could allow assembly of deadly virus DNA | Randerson<br />
; 2006 <sup>10</sup></a> </li><br />
<li>11 <a<br />
href="http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf">The<br />
Promise and Perils of Synthetic Biology | Tucker &amp; Zilinskas ; 2006<br />
<sup>11</sup></a></li><br />
<li>12 <a<br />
href="http://www.sciencemag.org/cgi/content/short/310/5745/17">1918<br />
Flu and Responsible Science | Sharp ; 2005 <sup>12</sup></a></li><br />
<li>13 <a href="http://www.fas.org/irp/cia/product/bw1103.pdf"> The<br />
Darker Bioweapons Future | CIA ; 2003 <sup>13</sup></a></li><br />
<li>14 <a href="http://openwetware.org/images/3/3d/SB_Primer_100707.pdf">Primer for Synthetic Biology | Mohr ; 2007 <sup>14</sup></a></li><br />
<li>15 [[Media:Freiburg10_The bugs of war.pdf]] </li><br />
<li>16 <a href="http://www.ncbi.nlm.nih.gov/pubmed/11152493">Expression<br />
of mouse interleukin-4 by a recombinant ectromelia virus suppresses<br />
cytolytic lymphocyte responses and overcomes genetic resistance to<br />
mousepox. | Jackson et al. ; 2001 <sup>16</sup></a></li><br />
<li>17 <a href="http://www.ncbi.nlm.nih.gov/pubmed/19784453">Governance<br />
of dual-use research: an ethical dilemma. | Selgelid ; 2009 <sup>17</sup></a></li><br />
<li>18 <a href="http://www.ncbi.nlm.nih.gov/pubmed/18625678">Evidence-based<br />
biosafety: a review of the principles and effectiveness of<br />
microbiological containment measures. | Kimman et al. 2008 <sup>18</sup></a><br />
</li><br />
<li>19 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819452">A<br />
Hippocratic Oath for life scientists | Revill et Dando ; 2006 <sup>19</sup></a></li><br />
<li>20 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819443">Empowerment<br />
and restraint in scientific communication. New developments make it<br />
easier to share information, but more difficult to deal with dual-use<br />
biology. | Campbell ; 2006<sup>20</sup></a></li><br />
<li>21 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819441">When<br />
risk<br />
outweighs benefit | Aken ; 2006 <sup>21</sup></a></li><br />
<li>22 <a href="http://www.ncbi.nlm.nih.gov/pubmed/12789408">Advances<br />
in life sciences and bioterrorism. Risks, perspectives and<br />
responsibilities.| Beck ; 2003 <sup>22</sup></a></li><br />
<li>23 <a href="http://www.ncbi.nlm.nih.gov/pubmed/12590130">PNAS<br />
policy on publication of sensitive material in the life sciences |<br />
Cozzarelli ; 2003 <sup>23</sup></a></li><br />
<li>24 <a<br />
href="http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf">Characterization<br />
of the<br />
Reconstructed 1918 Spanish<br />
Influenza Pandemic Virus | Tumpey et al. ; 2005<sup>24</sup></a> </li><br />
<li>25 <a<br />
href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf">Deadly<br />
flu virus can be<br />
sent through the mail| v. Bubnoff; 2005<sup>25</sup></a></li><br />
<li>26 <a<br />
href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf">Risk<br />
assessment of human Adeno-associated viruses| ZKBS; 2001<sup>26</sup></a></li><br />
<li>27 <a<br />
href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf">Advises<br />
for AAV carrying cell cycle regulating genes| ZKBS; 2004 <sup>27</sup></a></li><br />
<li>28 <a<br />
href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf">Risk<br />
assessment of human Adeno-associated viruses and AAV derived vectors|<br />
ZKBS; 2005 <sup>28</sup></a> </li><br />
<li>29 <a href="http://www.ncbi.nlm.nih.gov/pubmed/10700178">Evidence<br />
for gene transfer and expression of factor IX in haemophilia B patients<br />
treated with an AAV vector.| Kai et al. ; 2000 <sup>29</sup></a><br />
Bioverteilung in Klinischer Studie </li><br />
<li>30 <a<br />
href="https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf">Biosafety<br />
application of the iGEM team Freiburg_Bioware 2010<sup>30</sup></a> (in<br />
German)</li><br />
<li>31 <a<br />
href="https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg">Official<br />
classification as Biological Safety Level 1 by the local biosafety<br />
office<sup>31</sup></a></li><br />
<li>32 <a<br />
href="http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm">Appendix<br />
B | National Institute of Health <sup>32</sup></a></li><br />
<li>33 <a href="http://www.who.int/mediacentre/factsheets/smallpox/en"> <br />
Media centre - Smallpox | World Health Organisation <sup>33</sup></a></li><br />
<li>34 <a<br />
href="https://biotech.craic.com/blackwatch/introduction.html>BlackWatch Homepage | Craic computing<sup>34</sup></a></li><br />
<br />
<br />
<li>34 <a<br />
href="https://biotech.craic.com/blackwatch/introduction.html>BlackWatch Homepage | Craic computing<sup>34</sup></a></li><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</ul><br />
</html><br />
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<br />
<h1 style="text-align: justify;">Biosafety</h1><br />
<p style="text-align: justify;">Definition: "biosafety measures aim to<br />
prevent the unintentional<br />
exposure to pathogens and toxins, or their accidental release" <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br />
</p><br />
<h2 style="text-align: justify;">Risk assessment for our<br />
Adeno-associated virus based system</h2><br />
<p style="text-align: justify;">In Germany the "Central Commission for<br />
Biological Security" (ZKBS) released three legally binding Risk<br />
Assessment satements for the Adeno-associated Virus <sup>26,</sup><br />
<sup>27,</sup><br />
<sup>28</sup>.<br />
Risk assessment in other countries may deviate from these decisions, so<br />
please inform yourself about the legal regulations on AAV<br />
in your country before using the Virus Construction Kit.<br><br />
</p><br />
<div style="text-align: justify;">In Germany the Biological Safety<br />
Level(BSL) classifications for AAVs are:<br />
</div><br />
<ul style="text-align: justify;"><br />
<li>Adeno-associated Virus 2, 3 and 5 have to be handled under BSL 1.</li><br />
<li>Adeno-associated Viurs 1, 4, 5, 7, 8, 9, 10 and 11 have to be<br />
handled under BSL 2.</li><br />
</ul><br />
<p style="text-align: justify;">This classification was developed based<br />
on the fact that only serotypes 2, 3 and 5 are isolated from humans and<br />
that their harmlessness has been confirmed in clinical studies. This<br />
general classification has to be rechecked when the virus contains gene<br />
sequences with a transforming protential.<br />
</p><br />
<p style="text-align: justify;"><b>Viral vectors systems</b> packaging<br />
a vector plasmid that only contains the<br />
viral Inverted Terminal Repeats (ITRs) and providing the genes for<br />
Rep and Cap in trans (as it is the case for our system) are classified<br />
as <b>BSL 1</b> if the following conditions are fullfilled: </p><br />
<ul style="text-align: justify;"><br />
<li>The viral particles do not contain AAV derived sequences other<br />
than the ITRs</li><br />
<li>The viral particles do not contain Nucleotidesequences with a<br />
risk potential</li><br />
</ul><br />
<p style="text-align: justify;">We also investigated the legal<br />
regulations for AAV-2 Viral Vector<br />
systems in the United States. The guidelines of the National Institutes<br />
of Health (NIH) classify in the <a<br />
href="http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm">Appendix<br />
B</a><sup>32</sup> AAV-2 Vector Systems as Risk Group 1 (RG1) agents<br />
that can be treated under BSL 1. In detail the appendix states "...<br />
adeno- associated virus (AAV) types 1 through 4; and recombinant AAV<br />
constructs, in which the transgene does not encode either a potentially<br />
tumorigenic gene product or a toxin molecule and are produced in the<br />
absence of a helper virus."<br />
</p><br />
<div style="text-align: justify;">Other general observations with the<br />
Adeno-associated Virus 2 AAV-2)are: </div><br />
<ul style="text-align: justify;"><br />
<li>In clinical studies the viral vectors were not dilivered to the<br />
gonades. </li><br />
<li>Vector sequences were not detectable in the patients blood or<br />
urine at an examination 48 h after an infection with AAV-2 <sup>29</sup>.<br />
</li><br />
<li>In absence of Rep proteins, the vector DNA stays extrachromosomal<br />
and is not frequently integrated.</li><br />
</ul><br />
<p style="text-align: justify;"> Concluding all these informations and<br />
regulations, the project that we have designed in this year is clearly<br />
classified as BLS 1.<br />
</p><br />
<div style="text-align: justify;"> </div><br />
<h2 style="text-align: justify;">General biosafety regulation in Germany</h2><br />
<table<br />
style="text-align: left; width: 90%; margin-left: 0px; margin-right: 0px;"<br />
border="0" cellpadding="2" cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><br />
<p><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></p><br />
</td><br />
<td style="vertical-align: middle;"><span<br />
style="color: darkgreen;"><b>Which specific biosafety rules or<br />
guidelines do you have to consider in your country?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div style="text-align: justify;"><br />
</div><br />
<div<br />
style="margin: 0px 5px; width: 700px; float: right; height: auto; text-align: justify;"><span<br />
style="color: darkgreen;"> </span></div><br />
<div style="text-align: justify;"><br />
</div><br />
<p style="text-align: justify;">In Germany all work that includes<br />
recombinant DNA technologies is<br />
regulated by the <a href="http://bundesrecht.juris.de/gentg/index.html">Gesetz<br />
zur Regelung der Gentechnik</a>. This law regulates general<br />
aspects in the life sciences and refers for more precise<br />
interpretations in §4 to the <a<br />
href="http://bundesrecht.juris.de/gentg/__4.html">Zentrale<br />
Kommission für die Biologische Sicherheit</a>. The ZKBS is a<br />
commission composed of 20 technical experts that releases yearly<br />
statements to actual issues of biosafety. So far the ZKBS released<br />
three stratements affecting the work with Adeno-associated viral<br />
systems<br />
<a<br />
href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf"><sup>26,</sup></a><br />
<a<br />
href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf"><sup>27,</sup></a><br />
<a<br />
href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf"><sup>28</sup></a>.<br />
These documents were used to assess the dangers that could arise from<br />
our project to team members and the enviroment.<br />
</p><br />
<table<br />
style="text-align: left; width: 90%; margin-left: 0px; margin-right: 0px;"<br />
border="0" cellpadding="2" cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; width: 209px;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle; width: 778px;"><span<br />
style="color: DarkGreen;"><b>Is there a local biosafety group,<br />
committee, or review board at your institution? If yes, what does your<br />
local biosafety group think about your project?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><span<br />
style="color: DarkGreen;"></span></div><br />
<p style="text-align: justify;">At the Albert-Ludwigs-University<br />
Freiburg for all concerns of security the <a<br />
href="http://www.sicherheit.uni-freiburg.de">Stabsstelle Sicherheit</a><br />
is responsible and to contact if questions arise. Especially for<br />
questions of biological security Dr. Petra Markmeyer-Pieles is<br />
cognizant. We contacted her a first time befor the begin of our project<br />
in March when it was clear that the Adeno-associated Virus (AAV-2) was<br />
chosen as the topic of our project. At that time she proposed to do the<br />
cloning in the AAV-2 that is for sure to handle under biological<br />
security level 1 and to prepare everything for work under biological<br />
security level 2 to satisfy the precaution principle.<br />
The precaution principle was realized and all viral vectors that<br />
contained a modified capsid were handled under SII conditions until<br />
proven harmless.<br />
In August the planing of the project was completed, summarized in an <a<br />
href="https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf">Biosafety<br />
application<sup>30</sup></a> and handed to the department for<br />
biological security who approve the application in an <a<br />
href="https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg">official<br />
BSL1 confirmation<sup>31</sup></a>official BSL1 confirmation for our<br />
project.</p><br />
<h2>Risk management</h2><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: justify;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle; text-align: justify;"><span<br />
style="color: DarkGreen;"><b>Would any of your project ideas raise<br />
safety issues in terms of: researcher safety, public safety, or<br />
environmental safety?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p style="text-align: justify;"><br />
Our project was designed in a way that it avoids any serious safety<br />
issues as far as possible.<br />
When working with infectious particles a minimal risk for the<br />
researcher is allways present. This risk was minimized by restricting<br />
the transduced genes to fluorescent proteins and prodrug convertases<br />
that are already proven not to harm human cells in the absece of the<br />
corresponding prodrug. A potential danger for the public or the<br />
environment was minimized as much as possible by following strictly the<br />
rules of Good Laboratory Practice (GLP) and the abdication of using<br />
randomized insertions in the capsid and of replication potent viruses.<br />
Minimizing the risk for team members and the society was was allways<br />
one of the major concerns, especially because worries about<br />
undergraduate students manipulating a virus could arise.<br />
The security concept will be explained by quoting and explaining the<br />
six guiding principles for safe manipulation of Gene Manipulated<br />
Organisms (GMOs) as summarized in Kimman et al. ; 2008<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/18625678"><sup>18</sup></a>.<br />
</p><br />
<b>1) Hazard recognition and<br />
identification</b><br />
Risk assessment has been done and all legal regulations were considered<br />
as described in the last paragraph.<br />
<b><br><br />
2) Biological containment</b><br />
Biological containment means the usage of organisms with "reduced<br />
replicative capacity, inefectivity , transmissibility, and<br />
virulence"18.<br />
For our project only replicative deficient viruses were used,<br />
additional all modifications aimed to have a specific targeting of the<br />
resulting viral vectors for a specific cell type. This gain in<br />
specificity requires it to cut off the braod natural tropism resulting<br />
is an less infective virus copared to the wild type virus.<br />
<br><br />
<b>3) Concentration and enclosure</b><br />
All working steps for the preparation of viral vectors were carried out<br />
in a BSL II laboratory and within this laboratory work with the AAV was<br />
restricted to a separate Laminar flow cabinet type II. Cell culture and<br />
storage of the virus was also done in separate Freezers and incubators.<br />
All laboratories and epipment that contained viral vectors were<br />
specially marked, for example with a Biohazard warning signs.<br />
<b><br><br />
4) Exposure minimization</b><br />
This aspect of the guiding principles can be sumarized under "operator<br />
protection"18. The Exposure minimization was achieved in our laboratory<br />
by wearing special labcoats for the SII laboratory and gloves that were<br />
desinfected and changed regualrely. During manipulation of viral<br />
vectors attention was payed to avoid droplets and especially aerosoles.<br />
A possible diversion of the viral vectors was avoided by cleaning all<br />
equipment when inserted or removed from the Laminar flow cabinet and<br />
after completion of the work task.<br />
<b><br><br />
5) Physical containment</b><br />
The requirements for the physical containment were fullfilled by<br />
performing all manipulation on the AAV in an BSL II laboratory that<br />
guaranted a restriction of persons that entered the laboratory.<br />
<b><br><br />
6) Hazard minimization</b><br />
For the AAV-2 there are no sugestive activitis because the possible<br />
danger that runs out of the AAV is comparably low, vaccination is not<br />
avilible and biomonitoring is not necessary.<br />
<br><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Do any of the new BioBrick parts (or<br />
devices) that you made this year raise any safety issues? If yes, did<br />
you document these issues in the Registry? How did you manage to handle<br />
the safety issue? How could other teams learn from your experience?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p><br />
</p><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: justify;">Several<br />
composite parts that were assembled by our Team<br />
this year are alone capable of producing infectious viral particles<br />
when transduced together with a vector plasmid and a helper plasmid<br />
into <a<br />
href="https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf">AAV-293</a><br />
cells. These special cells provide the adenoviral gene E1 stabily<br />
integrated in trans.<br />
These cells are not provided in the Virus Construction Kit nor<br />
availible in the Parts Registry and have to purchased from other<br />
laboratories or a commercial supplyer.<br />
For this<br />
reason we estimate the risk of a accidental transformation of <a<br />
href="https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf">AAV-293</a><br />
cells with all three plasmids for negligible.<br />
Nevertheless we considered it useful to mark every BioBrick or<br />
Composite Part in the Registry that contributes to the production or is<br />
capable of producing viral vectors when transformed under the<br />
previously mentioned conditions.</td><br />
<td style="vertical-align: top;"><br />
<p><img margin:="" 0px="" 5px=""<br />
src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_Warning_SignI.png"<br />
alt="Warning sign for part descriptions" align="right" width="300"></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
</p><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Contribute to community discussions<br />
on what needs to go into a code against the use of our science for<br />
hostile purposes (see A Community Response)</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p><br />
</p><br />
<ul><br />
<li>Public Perception </li><br />
<li>importance of maintaining public legitimacy and support</li><br />
<li>apllications should demonstrate clear social benefits</li><br />
<li>not overhyped - anxiety and unrealistic hopes</li><br />
</ul><br />
<blockquote>psychological research into the concept of "identity-driven<br />
decision-making" (Torpman,2004)<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/16819452"> <sup>19</sup></a></blockquote><br />
<blockquote>Every grout has a set of norms: a code of conduct about<br />
what is acceptable beahviour (Jaques, 2004]<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/16819452"> <sup>19</sup></a></blockquote><br />
<br><br />
<br><br />
<center><br />
<h2>Trade-off between potential misuse and promising medical progress</h2><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: left;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Do you have any other ideas how to<br />
deal with safety issues that could be useful for future iGEM<br />
competitions? How could parts, devices and systems be made even safer<br />
through biosafety engineering?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
</center><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"> </span></div><br />
<p><br />
In principle each research-project that bears any risks for engaged<br />
researchers, mankind or the environment should be treated under the<br />
precautionary principle as proposed <a<br />
href="http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf"><sup>11</sup></a>:<br />
"treat synthetic microorganisms as dangerous until proven harmless".<br><br />
This would mean to work on such synthetic DNA containing Bio Bricks at<br />
least under Biological security levels two.<br><br />
Additional to this secure working environment the system itself can be<br />
optimized according to biosafety aspects, means to reduce it's<br />
viability outside the laboratory. This aim can be approached by<br />
reducing the systems ability to evolve, proliferate and interact with<br />
it's environment. A common method to achieve this goal is to engineer<br />
microorganisms in a way that they depend on nutrients that can't be<br />
found in the environment in sufficient amount.<br />
</p><br />
<br><br />
<br><br />
<center><br />
<h1>Biosecurity</h1><br />
</center><br />
Def: "measures focus on the prevention of theft, misuse , or<br />
intentional relese of pathogens and toxins" <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br><br />
<br><br />
<center><br />
<h2>The malignant use of biological agents in history</h2><br />
</center><br />
<p>The misuse of biological agent as weapons in warfare is a fear<br />
spreading companion in the history of mankind, ranging from the<br />
well-poisoners in prehistoric times to bio-terrorists present days. The<br />
following brakt intends to give a short outline of the major events<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a>:<br />
</p><br />
<ul><br />
<li> First systhematic use of chemical weapons during the first world<br />
war</li><br />
<li> The 1918 flu pandemic lasted from March 1918 to June 1920 and<br />
killed approximately 50 Million people around the world. Rumors<br />
circulated that this pandemic is caused by the other combatant nation.</li><br />
<li> "Prohibion of the Use of Asphyxiating, Poisonous or other Gases<br />
and of Bacteriological Methods of Warfare" was signed on 19 June 1925<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a></li><br />
<li> Several combatant nations of the second world war established<br />
biological warfare programs. </li><br />
<li> The range of the Japanese biological warfare program lead<br />
several<br />
nations to expand their own biological warfare program.</li><br />
<li> Limited military use<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a><br />
and the advances in molecular biology (e.g. the first isolatin of a<br />
gene and the discovery of the restriction enzymes in 1969) lead to the<br />
"Convention on the Prohibion of the Development, Production and<br />
Stockpilling of Bacteriological (Biological) and Toxin Weapons and on<br />
Their Destruction " (BTWC) on 10 April 1972.<br />
<blockquote>''"develop, produce, stockpile or otherwise acquire or<br />
retain: ... Microbial or other biological agents, or toxins whatever<br />
their origin or method of production, of types and in wantities that<br />
have no justification for prophylactic, protective or other peaceful<br />
purpose..." ''<sup>3</sup></blockquote><br />
</li><br />
<li> Breach of the BTWC by the Soviet Union which continued their<br />
offensive biologicla warfare programm </li><br />
<li> Stop of the biological warfare Program "Biopreparat" accompanies<br />
the Dissolution of the Svoviet Union in 1991</li><br />
<li> Antrax attacks in the USA in 2001</li><br />
After this short description of misused biological components there is<br />
still the question what impact biological warfare will have in the<br />
future of mankind.<br><br />
<br><br />
</ul><br />
<center><br />
<h2>Broad avilibility of knowledge</h2><br />
</center><br />
n the life sciences information has allways been freely acessible for<br />
everybody who is interested in the results of a particular research<br />
project. The combination of this global availibility with the new<br />
possibilities of the internet broad for literature search and<br />
availibility of gene sequences made it easiere to collect informations<br />
for a possible misuse. <br><br />
For the overwhelming majority this open availibility is absolutely<br />
desirely but on the other hand there are also examples of research<br />
results that bear a very high risk to be misused. <br><br />
Following we present the three most controversial discussed<br />
publications that could also be read as a "How to create your own<br />
bioweapon".<br />
<h3>Mousepox Virus in Australien</h3><br />
<br><br />
The Australian research group around <a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/11152493"> Jackson et al. ;<br />
2001 <sup>16</sup></a>inserted the coding region of the IL-4 gne into<br />
the genome of mousepox. They hoped to create a virus that sterilizes<br />
mice and thus provides a means for pest control. Contrarely to their<br />
expectations they had created a superstrain that killed even naturally<br />
resistent mice and mice that had been vaccinated against normal<br />
mousepox. This discovery could potentially be used to make smallpox<br />
resistant to potential vaccines.<br />
<h3>Synthesis of a Polio Virus</h3><br />
<br><br />
At the State University of New York <a<br />
href="http://www.sciencemag.org/cgi/content/full/297/5583/1016"> Cello<br />
et al. ; 2002 <sup>05</sup> synthesized a "living" polio virus from<br />
scratch. Especially the </a><a<br />
href="http://www.sciencemag.org/cgi/content/full/1072266/DC1">Supporting<br />
Online Material</a> caused worried remarks because it precisely<br />
describes how to assemble a virus from small oligo nucleotides. The<br />
authors jusifyed their project by declaring that they:<br />
<blockquote>"made the virus to send a warning that terrorists might be<br />
able to make biological weapons without obtaining a natrual virus"<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/19784453"><sup>17</sup></a></blockquote><br />
<h3>Reconstitution of the Spanish Flu</h3><br />
<br><br />
<img style="width: 300px; height: 223px;" class="right"<br />
src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Spanishflu.jpg"<br />
alt="Influenza hospital for american soldiers" align="left"><br />
At the Center for Disease Control and Prevention (CDC) the group around<br />
<a href="http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf">Tumpey<br />
et al. ; 2005<sup>24</sup></a> published that they had sequenced and<br />
recreated the pandemic Spanish Flu Virus of 1918 which killed 20-50<br />
million people. The publication of the sequence provoked several very<br />
different responses that can only be partially be quoted here.<br />
The controversial noticed outrider of the Synthetic Biology Craig<br />
Venter seen in the new virus<br />
<blockquote>"the first true Juressic Parc scenario" <a<br />
href="http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&amp;sID=4233"><sup>2</sup></a></blockquote><br />
The Institute Professor at the MIT Philip A. Sharp supported the<br />
publication because he:<br />
<blockquote>"belive[s] that allowing the publication of this<br />
information was the correct decision in terms of both national security<br />
and public health" <a<br />
href="http://www.sciencemag.org/cgi/content/short/310/5745/17"><sup>12</sup></a></blockquote><br />
Wheras <a<br />
href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf">v.<br />
Bubnoff; 2005<sup>25</sup></a> critisizes the CDS for it's careless<br />
regulations for the shipment of viruses and the willingness to<br />
propagate highly virulent viruses. This causes in his opinion the risk<br />
of possible accidents resulting in the release of the virus to the<br />
environment.<br><br />
<br><br />
<h3>Availability of molecular biological techniques</h3><br />
<br />
The knowledge required for the creation of a genetically engineered virus can easily be accessed from all over the world in online gene banks and publication databases and could be potentially be used for a destructive purpose <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819443"><sup>20</sup></a>. In 2007, Scott C. Mohr published the first part of his manuscript <a href="http://openwetware.org/images/3/3d/SB_Primer_100707.pdf">Primer for Synthetic Biology<sup>14</sup></a></li>. This open accessible document explaining the basics of molecular engineering addresses people interested in genetics but coming from a non-academic background. This development called "garage biology" or "biohacking" is a clear indication for the tendency to have knowledge about molecular biology available in society, as it was seen with programming and computer hacking from ~1980 on.<br />
<br />
<h3>Availability of synthesized DNA</h3><br />
<br />
<br />
One of the reactions to the publication of the genome sequence of the Spanish flu strain from 1918 was <a href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf"> v. Bubnoff, 2005<sup>25</sup></a></li> who pointed out the lax handling of the reconstituted virus sequence, the ease of availability and the low effort that has to be invested to recreate a virus:<br />
<blockquote>''"Scientists in Canada are planning to work with the virus, although they will not request it from the CDC. [...] Reconstructing the live virus from its DNA would then take just a few days, he says.''</blockquote><br />
Technical advances and dropping prices in the field of gene synthesis brings several deadly germs into the range of small research projects or even private persons as for example Spanish Flu (Influenza H<sub>1</sub>N<sub>1</sub>) with a 13.5 kbp <a href=http://expasy.org/viralzone/all_by_species/131.html><sup>*</sup></a> or even the Ebola virus with a 19 kbp genome <a href=http://expasy.org/viralzone/all_by_species/207.html><sup>*</sup></a>.<br />
<br />
In 2006, the “Guardian” journalist James Randson ordered a 78 bp DNA oligonucleotide coding for the capsid of the smallpox <i>Orthopoxvirus variola</i> with the intention to alert the public.<br />
<a href="http://www.guardian.co.uk/world/2006/jun/14/terrorism.topstories3"><sup>10</sup></a> <br><br />
In the United States, smallpox are listed as schedule 5 pathogens in the <i>Anti-terrorism, Crime and Security Act of 2001</i> and are illegal to keep or use without first notifying the civil authorities.<br><br />
On the other hand, it is questionable if these prohibitions or perhaps the size of the smallpox genome of ~200 kb <a href=http://expasy.org/viralzone/all_by_species/149.html><sup>*</sup></a> are sufficient to prevent de novo synthesis in the future efficiently. His intention was to focus the public's attention to the possibility that one of the most severe plagues in the history of mankind could be synthesized and released to the environment, either intentionally or by mistake. This would be a tragic setback since the smallpox's eradication was celebrated in 1979 by the World Health Organization as one of the greatest victories in the history of medicine <a href=http://www.who.int/mediacentre/factsheets/smallpox/en><sup>33</sup></a>.<br><br />
In order to prevent the unauthorized synthesis of gene sequences encoding hazardous biological agents, efforts to implement sequence analysis algorithms into the operating procedure of all organizations and companies capable of synthesizing gene sequences are being undertaken.<br> <br><br />
This barrier should be implemented not on a voluntary basis but as a legally binding regulation. It should be enforced by the government on a national level, but effort should also be made to find solutions for an international progress on this issue.<br><br />
As a first step, the development of search algorithms as Craic's BlackWatch<a<br />
href="https://biotech.craic.com/blackwatch/introduction.html><sup>34</sup></a> should be promoted and refined.<br><br><br />
Additional to this sequence base search for possible misuse of gene sequences, each order of already existing or synthesized genes could first be aligned with a list of countries, and in a second search with the so called Hadex exclusion list that names people and organizations excluded from obtaining dual-use gene material<a href=http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf><sup>1</sup></a>. <br />
<br><br />
<h2>The nature of biological weapons - Who is willing to accept their drawbacks</h2><br />
When considering the use of biological means for warfare, a potential aggressor has to accept several serious drawbacks. Biological weapons are not fully controllable, harmful effects to the civilian population cannot be foreseen. If replication-potent germs were used for such a purpose, they could possibly mutate and seriously harm mankind or other animal populations.<br><br />
This makes biological warfare unattractive for most nations, especially because more predictable weapons exist that can easily be directed against military targets with limited collateral damage.<br />
Unfortunately, national warfare programs are not the only source of danger when considering biological warfare. <br />
<blockquote>"An increasing number of countries believe that their political and security interests could be protected or achieved only through the possession of such weapons, especially in view of the overwhelming superiority of the US armed forces in terms of conventional weapons."<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a></blockquote> <br />
Several countries around the globe are technically capable to develop biological weapons or to support terroristic groups in doing so. When this physical capability meets an ideological attitude that is based on contempt for other concepts of society, it cannot be ruled out that biological warfare is taken into consideration. <br><br />
<br />
<h2>Synthetic Biology vs. Nuclear Research - Consideration in terms of security policy<h3> <br />
When considering the history of research, the dual-use character of technical innovations has always been present, only the reaction of society to it differs from case to case. As an example, scientific discoveries in the field of nuclear physics where treated as classified information having a high relevance for the national security as shown in the case of the Manhattan project<a href="http://www.ncbi.nlm.nih.gov/pubmed/19784453"><sup>17</sup></a>. A comparable censorship is not established in life sciences research apart of some cases when national security was endangered.<br><br><br />
Another point concerning the threat arising from biological weapons is the absence of methods that can be used to monitor such weapons of mass destruction as pointed out by M.R. Dando. This fact and the increasing world-wide mobility would make it impossible to prevent the spreading of such weapons when once accessible to potential assassins. <br />
<br />
<h3>Is a revision of basic research necessary in sensitive research fields? </h3><br />
The publication of research papers containing dual-use knowledge caused several people to call for regulation. In the United States, the so called Fink Committee evaluated the possibility that research in life sciences could be used for biological warfare purposes and how this could be avoided.<a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br><br><br />
<br />
This committee had two recommendations:<br />
<ul><br />
<li>To familiarize the scientific community with the dual-use problem in the life sciences.</li><br />
<li>To review experiments of concern. Experiments are of concern if they:<br />
<ul><br />
<li>render a vaccine ineffective</li><br />
<li>confer resistance to therapeutically useful antibiotics or<br />
antiviral agents</li><br />
<li>enhance the virulence of a pathogen or render a nonpathogen<br />
virulent</li><br />
<li>increase transmissibility of a pathogen</li><br />
<li>alter the host range of a pathogen</li><br />
<li>enable the evasion of diagnostic/detection tools</li><br />
<li>enable the weaponization of a biological agent or toxin</li></ul><br />
<li>To review publications with a strong dual-use factor</li><br />
<li>To create a National Science Advisory Board</li><br />
<li>To improve the oversight and reduce unauthorized accessibility of hazardous gene material</li><br />
<li>To include the life sciences into the efforts for national security</li><br />
<li>To harmonize the international oversight over dual-use research</li><br />
</ul><br />
<br />
The implementation of these recommendations would be desirable, even though they might cause inconveniences for scientists working in the affected fields.<br />
<br />
<br />
<br />
<br />
<h2>Conclusion</h2><br />
<br />
<br />
Concluding all factors mentioned above, we need to consider the possibility that a person, organization or state could misuse the fast-advancing life sciences for biological warfare. The possibilities and the simplicity of dual-use research misuse will become more and easier the faster the scientific progress advances.<br><br />
Therefore it is important to minimize these potential risks before they become reality. This is not only the task of a designated group but a moral obligation for scientists, politicians and everybody related to dual-use research. Especially scientists have to contribute to and lead the continuing discussion on this topic because they are able to estimate how aspects of their research fields might be misused.<br />
<br />
<center><br />
<h2>Conclusion</h2><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Include<br />
something in your project<br />
description and presentations that demonstrates that you have thought<br />
about how others could misuse your work.</b></span> </td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
</center><br />
<p>For sure there is allway the possibility that knowledge to produce<br />
transgene viral vectors could be used to produce bioweapons. Therefor<br />
it was important for us to use a system that does not bear the risk<br />
that someone could use it for evil purpose. In the case of the<br />
Adeno-associated virus the very limited packaging capacity is the major<br />
reason that excludes it from the list of agents that could<br />
realistically be used for the pruduction of bioweapons. Even a fully<br />
replication potent AAV will depend on the coninfection of a helpervirus<br />
and is therefore not suitable for a fast propagation in an population.<br />
Additional to this point we concentrated our project on the retargeting<br />
of the virus - means to make the broad tropismn more narrow and to<br />
decrease the transduction efficiency in the most cases. This<br />
modification is usually mainly required for medical purposes. Also we<br />
did neither investigate possibilities to shield the vector from the<br />
immune system of potential host nor ways to bypass an existing<br />
immunity. </p><br />
<br><br />
<br><br />
<ul><br />
<li>01 <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf">Synthetic<br />
Biology &amp; Biosecurity - Awareness in Europe | Kelle ; 2007<sup>1</sup></a></li><br />
<li>02 <a<br />
href="http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&amp;sID=4233">Synthetic<br />
Biology - Social and Ethical Challenges | Balmer et Martin ; 2008<sup>2</sup></a></li><br />
<li>03 Synthetic Biology - Applying Engineering to Biology <br><br />
used </li><br />
<li>04 <a<br />
href="http://www.informaworld.com/smpp/content%7Econtent=a713604665&amp;db=all">The<br />
Impact of the Development of Modern Biology and Medicine on the<br />
Evolution of Offensive Biological Warfare Programs in the Twentieth<br />
Century | Dando ; 1999 <sup>04</sup></a></li><br />
<li>05 <a<br />
href="http://www.sciencemag.org/cgi/content/full/297/5583/1016">Chemical<br />
Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the<br />
Absence of Natural Template | Cello et al. ; 2002 <sup>05</sup></a></li><br />
<li><a href="http://www.sciencemag.org/cgi/content/full/297/5583/1016">06<br />
</a><a<br />
href="http://www.nytimes.com/2003/12/03/opinion/03iht-edstein_ed3_.html">Dangerous<br />
research : When science breeds nightmares | Steinbruner et Harris ;<br />
2003 <sup>06</sup></a></li><br />
<li>07 <a<br />
href="http://www.jcvi.org/cms/fileadmin/site/research/projects/synthetic-genomics-report/synthetic-genomics-report.pdf">Synthetic<br />
Genomics - Options for governance | Garfinkel et al. ; 2007 <sup>07</sup></a></li><br />
<li>08 <a<br />
href="http://www.nature.com/nrg/journal/v6/n7/abs/nrg1637.html">Synthetic<br />
biology | Benner et Sismour , 2005 <sup>08</sup></a></li><br />
<li>09 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16954140">Synthetic<br />
biology—putting engineering into biology | Heinemann et Panke ; 2006 <sup>09</sup></a></li><br />
<li>10 <a<br />
href="http://www.guardian.co.uk/world/2006/jun/14/terrorism.topstories3">Revealed:<br />
the lax laws that could allow assembly of deadly virus DNA | Randerson<br />
; 2006 <sup>10</sup></a> </li><br />
<li>11 <a<br />
href="http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf">The<br />
Promise and Perils of Synthetic Biology | Tucker &amp; Zilinskas ; 2006<br />
<sup>11</sup></a></li><br />
<li>12 <a<br />
href="http://www.sciencemag.org/cgi/content/short/310/5745/17">1918<br />
Flu and Responsible Science | Sharp ; 2005 <sup>12</sup></a></li><br />
<li>13 <a href="http://www.fas.org/irp/cia/product/bw1103.pdf"> The<br />
Darker Bioweapons Future | CIA ; 2003 <sup>13</sup></a></li><br />
<li>14 <a href="http://openwetware.org/images/3/3d/SB_Primer_100707.pdf">Primer for Synthetic Biology | Mohr ; 2007 <sup>14</sup></a></li><br />
<li>15 [[Media:Freiburg10_The bugs of war.pdf]] </li><br />
<li>16 <a href="http://www.ncbi.nlm.nih.gov/pubmed/11152493">Expression<br />
of mouse interleukin-4 by a recombinant ectromelia virus suppresses<br />
cytolytic lymphocyte responses and overcomes genetic resistance to<br />
mousepox. | Jackson et al. ; 2001 <sup>16</sup></a></li><br />
<li>17 <a href="http://www.ncbi.nlm.nih.gov/pubmed/19784453">Governance<br />
of dual-use research: an ethical dilemma. | Selgelid ; 2009 <sup>17</sup></a></li><br />
<li>18 <a href="http://www.ncbi.nlm.nih.gov/pubmed/18625678">Evidence-based<br />
biosafety: a review of the principles and effectiveness of<br />
microbiological containment measures. | Kimman et al. 2008 <sup>18</sup></a><br />
</li><br />
<li>19 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819452">A<br />
Hippocratic Oath for life scientists | Revill et Dando ; 2006 <sup>19</sup></a></li><br />
<li>20 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819443">Empowerment<br />
and restraint in scientific communication. New developments make it<br />
easier to share information, but more difficult to deal with dual-use<br />
biology. | Campbell ; 2006<sup>20</sup></a></li><br />
<li>21 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819441">When<br />
risk<br />
outweighs benefit | Aken ; 2006 <sup>21</sup></a></li><br />
<li>22 <a href="http://www.ncbi.nlm.nih.gov/pubmed/12789408">Advances<br />
in life sciences and bioterrorism. Risks, perspectives and<br />
responsibilities.| Beck ; 2003 <sup>22</sup></a></li><br />
<li>23 <a href="http://www.ncbi.nlm.nih.gov/pubmed/12590130">PNAS<br />
policy on publication of sensitive material in the life sciences |<br />
Cozzarelli ; 2003 <sup>23</sup></a></li><br />
<li>24 <a<br />
href="http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf">Characterization<br />
of the<br />
Reconstructed 1918 Spanish<br />
Influenza Pandemic Virus | Tumpey et al. ; 2005<sup>24</sup></a> </li><br />
<li>25 <a<br />
href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf">Deadly<br />
flu virus can be<br />
sent through the mail| v. Bubnoff; 2005<sup>25</sup></a></li><br />
<li>26 <a<br />
href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf">Risk<br />
assessment of human Adeno-associated viruses| ZKBS; 2001<sup>26</sup></a></li><br />
<li>27 <a<br />
href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf">Advises<br />
for AAV carrying cell cycle regulating genes| ZKBS; 2004 <sup>27</sup></a></li><br />
<li>28 <a<br />
href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf">Risk<br />
assessment of human Adeno-associated viruses and AAV derived vectors|<br />
ZKBS; 2005 <sup>28</sup></a> </li><br />
<li>29 <a href="http://www.ncbi.nlm.nih.gov/pubmed/10700178">Evidence<br />
for gene transfer and expression of factor IX in haemophilia B patients<br />
treated with an AAV vector.| Kai et al. ; 2000 <sup>29</sup></a><br />
Bioverteilung in Klinischer Studie </li><br />
<li>30 <a<br />
href="https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf">Biosafety<br />
application of the iGEM team Freiburg_Bioware 2010<sup>30</sup></a> (in<br />
German)</li><br />
<li>31 <a<br />
href="https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg">Official<br />
classification as Biological Safety Level 1 by the local biosafety<br />
office<sup>31</sup></a></li><br />
<li>32 <a<br />
href="http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm">Appendix<br />
B | National Institute of Health <sup>32</sup></a></li><br />
<li>33 <a href="http://www.who.int/mediacentre/factsheets/smallpox/en"> <br />
Media centre - Smallpox | World Health Organisation <sup>33</sup></a></li><br />
<li>34 <a<br />
href="https://biotech.craic.com/blackwatch/introduction.html>BlackWatch Homepage | Craic computing<sup>34</sup></a></li><br />
<br />
<br />
<li>34 <a<br />
href="https://biotech.craic.com/blackwatch/introduction.html>BlackWatch Homepage | Craic computing<sup>34</sup></a></li><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</ul><br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/SafetyTeam:Freiburg Bioware/Safety2010-10-28T03:18:19Z<p>Achim: </p>
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<br />
<h1 style="text-align: justify;">Biosafety</h1><br />
<p style="text-align: justify;">Definition: "biosafety measures aim to<br />
prevent the unintentional<br />
exposure to pathogens and toxins, or their accidental release" <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br />
</p><br />
<h2 style="text-align: justify;">Risk assessment for our<br />
Adeno-associated virus based system</h2><br />
<p style="text-align: justify;">In Germany the "Central Commission for<br />
Biological Security" (ZKBS) released three legally binding Risk<br />
Assessment satements for the Adeno-associated Virus <sup>26,</sup><br />
<sup>27,</sup><br />
<sup>28</sup>.<br />
Risk assessment in other countries may deviate from these decisions, so<br />
please inform yourself about the legal regulations on AAV<br />
in your country before using the Virus Construction Kit.<br><br />
</p><br />
<div style="text-align: justify;">In Germany the Biological Safety<br />
Level(BSL) classifications for AAVs are:<br />
</div><br />
<ul style="text-align: justify;"><br />
<li>Adeno-associated Virus 2, 3 and 5 have to be handled under BSL 1.</li><br />
<li>Adeno-associated Viurs 1, 4, 5, 7, 8, 9, 10 and 11 have to be<br />
handled under BSL 2.</li><br />
</ul><br />
<p style="text-align: justify;">This classification was developed based<br />
on the fact that only serotypes 2, 3 and 5 are isolated from humans and<br />
that their harmlessness has been confirmed in clinical studies. This<br />
general classification has to be rechecked when the virus contains gene<br />
sequences with a transforming protential.<br />
</p><br />
<p style="text-align: justify;"><b>Viral vectors systems</b> packaging<br />
a vector plasmid that only contains the<br />
viral Inverted Terminal Repeats (ITRs) and providing the genes for<br />
Rep and Cap in trans (as it is the case for our system) are classified<br />
as <b>BSL 1</b> if the following conditions are fullfilled: </p><br />
<ul style="text-align: justify;"><br />
<li>The viral particles do not contain AAV derived sequences other<br />
than the ITRs</li><br />
<li>The viral particles do not contain Nucleotidesequences with a<br />
risk potential</li><br />
</ul><br />
<p style="text-align: justify;">We also investigated the legal<br />
regulations for AAV-2 Viral Vector<br />
systems in the United States. The guidelines of the National Institutes<br />
of Health (NIH) classify in the <a<br />
href="http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm">Appendix<br />
B</a><sup>32</sup> AAV-2 Vector Systems as Risk Group 1 (RG1) agents<br />
that can be treated under BSL 1. In detail the appendix states "...<br />
adeno- associated virus (AAV) types 1 through 4; and recombinant AAV<br />
constructs, in which the transgene does not encode either a potentially<br />
tumorigenic gene product or a toxin molecule and are produced in the<br />
absence of a helper virus."<br />
</p><br />
<div style="text-align: justify;">Other general observations with the<br />
Adeno-associated Virus 2 AAV-2)are: </div><br />
<ul style="text-align: justify;"><br />
<li>In clinical studies the viral vectors were not dilivered to the<br />
gonades. </li><br />
<li>Vector sequences were not detectable in the patients blood or<br />
urine at an examination 48 h after an infection with AAV-2 <sup>29</sup>.<br />
</li><br />
<li>In absence of Rep proteins, the vector DNA stays extrachromosomal<br />
and is not frequently integrated.</li><br />
</ul><br />
<p style="text-align: justify;"> Concluding all these informations and<br />
regulations, the project that we have designed in this year is clearly<br />
classified as BLS 1.<br />
</p><br />
<div style="text-align: justify;"> </div><br />
<h2 style="text-align: justify;">General biosafety regulation in Germany</h2><br />
<table<br />
style="text-align: left; width: 90%; margin-left: 0px; margin-right: 0px;"<br />
border="0" cellpadding="2" cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><br />
<p><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></p><br />
</td><br />
<td style="vertical-align: middle;"><span<br />
style="color: darkgreen;"><b>Which specific biosafety rules or<br />
guidelines do you have to consider in your country?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div style="text-align: justify;"><br />
</div><br />
<div<br />
style="margin: 0px 5px; width: 700px; float: right; height: auto; text-align: justify;"><span<br />
style="color: darkgreen;"> </span></div><br />
<div style="text-align: justify;"><br />
</div><br />
<p style="text-align: justify;">In Germany all work that includes<br />
recombinant DNA technologies is<br />
regulated by the <a href="http://bundesrecht.juris.de/gentg/index.html">Gesetz<br />
zur Regelung der Gentechnik</a>. This law regulates general<br />
aspects in the life sciences and refers for more precise<br />
interpretations in §4 to the <a<br />
href="http://bundesrecht.juris.de/gentg/__4.html">Zentrale<br />
Kommission für die Biologische Sicherheit</a>. The ZKBS is a<br />
commission composed of 20 technical experts that releases yearly<br />
statements to actual issues of biosafety. So far the ZKBS released<br />
three stratements affecting the work with Adeno-associated viral<br />
systems<br />
<a<br />
href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf"><sup>26,</sup></a><br />
<a<br />
href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf"><sup>27,</sup></a><br />
<a<br />
href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf"><sup>28</sup></a>.<br />
These documents were used to assess the dangers that could arise from<br />
our project to team members and the enviroment.<br />
</p><br />
<table<br />
style="text-align: left; width: 90%; margin-left: 0px; margin-right: 0px;"<br />
border="0" cellpadding="2" cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; width: 209px;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle; width: 778px;"><span<br />
style="color: DarkGreen;"><b>Is there a local biosafety group,<br />
committee, or review board at your institution? If yes, what does your<br />
local biosafety group think about your project?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><span<br />
style="color: DarkGreen;"></span></div><br />
<p style="text-align: justify;">At the Albert-Ludwigs-University<br />
Freiburg for all concerns of security the <a<br />
href="http://www.sicherheit.uni-freiburg.de">Stabsstelle Sicherheit</a><br />
is responsible and to contact if questions arise. Especially for<br />
questions of biological security Dr. Petra Markmeyer-Pieles is<br />
cognizant. We contacted her a first time befor the begin of our project<br />
in March when it was clear that the Adeno-associated Virus (AAV-2) was<br />
chosen as the topic of our project. At that time she proposed to do the<br />
cloning in the AAV-2 that is for sure to handle under biological<br />
security level 1 and to prepare everything for work under biological<br />
security level 2 to satisfy the precaution principle.<br />
The precaution principle was realized and all viral vectors that<br />
contained a modified capsid were handled under SII conditions until<br />
proven harmless.<br />
In August the planing of the project was completed, summarized in an <a<br />
href="https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf">Biosafety<br />
application<sup>30</sup></a> and handed to the department for<br />
biological security who approve the application in an <a<br />
href="https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg">official<br />
BSL1 confirmation<sup>31</sup></a>official BSL1 confirmation for our<br />
project.</p><br />
<h2>Risk management</h2><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: justify;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle; text-align: justify;"><span<br />
style="color: DarkGreen;"><b>Would any of your project ideas raise<br />
safety issues in terms of: researcher safety, public safety, or<br />
environmental safety?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p style="text-align: justify;"><br />
Our project was designed in a way that it avoids any serious safety<br />
issues as far as possible.<br />
When working with infectious particles a minimal risk for the<br />
researcher is allways present. This risk was minimized by restricting<br />
the transduced genes to fluorescent proteins and prodrug convertases<br />
that are already proven not to harm human cells in the absece of the<br />
corresponding prodrug. A potential danger for the public or the<br />
environment was minimized as much as possible by following strictly the<br />
rules of Good Laboratory Practice (GLP) and the abdication of using<br />
randomized insertions in the capsid and of replication potent viruses.<br />
Minimizing the risk for team members and the society was was allways<br />
one of the major concerns, especially because worries about<br />
undergraduate students manipulating a virus could arise.<br />
The security concept will be explained by quoting and explaining the<br />
six guiding principles for safe manipulation of Gene Manipulated<br />
Organisms (GMOs) as summarized in Kimman et al. ; 2008<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/18625678"><sup>18</sup></a>.<br />
</p><br />
<b>1) Hazard recognition and<br />
identification</b><br />
Risk assessment has been done and all legal regulations were considered<br />
as described in the last paragraph.<br />
<b><br><br />
2) Biological containment</b><br />
Biological containment means the usage of organisms with "reduced<br />
replicative capacity, inefectivity , transmissibility, and<br />
virulence"18.<br />
For our project only replicative deficient viruses were used,<br />
additional all modifications aimed to have a specific targeting of the<br />
resulting viral vectors for a specific cell type. This gain in<br />
specificity requires it to cut off the braod natural tropism resulting<br />
is an less infective virus copared to the wild type virus.<br />
<br><br />
<b>3) Concentration and enclosure</b><br />
All working steps for the preparation of viral vectors were carried out<br />
in a BSL II laboratory and within this laboratory work with the AAV was<br />
restricted to a separate Laminar flow cabinet type II. Cell culture and<br />
storage of the virus was also done in separate Freezers and incubators.<br />
All laboratories and epipment that contained viral vectors were<br />
specially marked, for example with a Biohazard warning signs.<br />
<b><br><br />
4) Exposure minimization</b><br />
This aspect of the guiding principles can be sumarized under "operator<br />
protection"18. The Exposure minimization was achieved in our laboratory<br />
by wearing special labcoats for the SII laboratory and gloves that were<br />
desinfected and changed regualrely. During manipulation of viral<br />
vectors attention was payed to avoid droplets and especially aerosoles.<br />
A possible diversion of the viral vectors was avoided by cleaning all<br />
equipment when inserted or removed from the Laminar flow cabinet and<br />
after completion of the work task.<br />
<b><br><br />
5) Physical containment</b><br />
The requirements for the physical containment were fullfilled by<br />
performing all manipulation on the AAV in an BSL II laboratory that<br />
guaranted a restriction of persons that entered the laboratory.<br />
<b><br><br />
6) Hazard minimization</b><br />
For the AAV-2 there are no sugestive activitis because the possible<br />
danger that runs out of the AAV is comparably low, vaccination is not<br />
avilible and biomonitoring is not necessary.<br />
<br><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Do any of the new BioBrick parts (or<br />
devices) that you made this year raise any safety issues? If yes, did<br />
you document these issues in the Registry? How did you manage to handle<br />
the safety issue? How could other teams learn from your experience?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p><br />
</p><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: justify;">Several<br />
composite parts that were assembled by our Team<br />
this year are alone capable of producing infectious viral particles<br />
when transduced together with a vector plasmid and a helper plasmid<br />
into <a<br />
href="https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf">AAV-293</a><br />
cells. These special cells provide the adenoviral gene E1 stabily<br />
integrated in trans.<br />
These cells are not provided in the Virus Construction Kit nor<br />
availible in the Parts Registry and have to purchased from other<br />
laboratories or a commercial supplyer.<br />
For this<br />
reason we estimate the risk of a accidental transformation of <a<br />
href="https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf">AAV-293</a><br />
cells with all three plasmids for negligible.<br />
Nevertheless we considered it useful to mark every BioBrick or<br />
Composite Part in the Registry that contributes to the production or is<br />
capable of producing viral vectors when transformed under the<br />
previously mentioned conditions.</td><br />
<td style="vertical-align: top;"><br />
<p><img margin:="" 0px="" 5px=""<br />
src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_Warning_SignI.png"<br />
alt="Warning sign for part descriptions" align="right" width="300"></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
</p><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Contribute to community discussions<br />
on what needs to go into a code against the use of our science for<br />
hostile purposes (see A Community Response)</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"></span></div><br />
<p><br />
</p><br />
<ul><br />
<li>Public Perception </li><br />
<li>importance of maintaining public legitimacy and support</li><br />
<li>apllications should demonstrate clear social benefits</li><br />
<li>not overhyped - anxiety and unrealistic hopes</li><br />
</ul><br />
<blockquote>psychological research into the concept of "identity-driven<br />
decision-making" (Torpman,2004)<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/16819452"> <sup>19</sup></a></blockquote><br />
<blockquote>Every grout has a set of norms: a code of conduct about<br />
what is acceptable beahviour (Jaques, 2004]<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/16819452"> <sup>19</sup></a></blockquote><br />
<br><br />
<br><br />
<center><br />
<h2>Trade-off between potential misuse and promising medical progress</h2><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top; text-align: left;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Do you have any other ideas how to<br />
deal with safety issues that could be useful for future iGEM<br />
competitions? How could parts, devices and systems be made even safer<br />
through biosafety engineering?</b></span></td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
</center><br />
<div<br />
style="margin: 0px 5px; float: right; width: 700px; height: auto; text-align: justify;"><br />
<span style="color: DarkGreen;"> </span></div><br />
<p><br />
In principle each research-project that bears any risks for engaged<br />
researchers, mankind or the environment should be treated under the<br />
precautionary principle as proposed <a<br />
href="http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf"><sup>11</sup></a>:<br />
"treat synthetic microorganisms as dangerous until proven harmless".<br><br />
This would mean to work on such synthetic DNA containing Bio Bricks at<br />
least under Biological security levels two.<br><br />
Additional to this secure working environment the system itself can be<br />
optimized according to biosafety aspects, means to reduce it's<br />
viability outside the laboratory. This aim can be approached by<br />
reducing the systems ability to evolve, proliferate and interact with<br />
it's environment. A common method to achieve this goal is to engineer<br />
microorganisms in a way that they depend on nutrients that can't be<br />
found in the environment in sufficient amount.<br />
</p><br />
<br><br />
<br><br />
<center><br />
<h1>Biosecurity</h1><br />
</center><br />
Def: "measures focus on the prevention of theft, misuse , or<br />
intentional relese of pathogens and toxins" <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br><br />
<br><br />
<center><br />
<h2>The malignant use of biological agents in history</h2><br />
</center><br />
<p>The misuse of biological agent as weapons in warfare is a fear<br />
spreading companion in the history of mankind, ranging from the<br />
well-poisoners in prehistoric times to bio-terrorists present days. The<br />
following brakt intends to give a short outline of the major events<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a>:<br />
</p><br />
<ul><br />
<li> First systhematic use of chemical weapons during the first world<br />
war</li><br />
<li> The 1918 flu pandemic lasted from March 1918 to June 1920 and<br />
killed approximately 50 Million people around the world. Rumors<br />
circulated that this pandemic is caused by the other combatant nation.</li><br />
<li> "Prohibion of the Use of Asphyxiating, Poisonous or other Gases<br />
and of Bacteriological Methods of Warfare" was signed on 19 June 1925<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a></li><br />
<li> Several combatant nations of the second world war established<br />
biological warfare programs. </li><br />
<li> The range of the Japanese biological warfare program lead<br />
several<br />
nations to expand their own biological warfare program.</li><br />
<li> Limited military use<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/12789408"><sup>22</sup></a><br />
and the advances in molecular biology (e.g. the first isolatin of a<br />
gene and the discovery of the restriction enzymes in 1969) lead to the<br />
"Convention on the Prohibion of the Development, Production and<br />
Stockpilling of Bacteriological (Biological) and Toxin Weapons and on<br />
Their Destruction " (BTWC) on 10 April 1972.<br />
<blockquote>''"develop, produce, stockpile or otherwise acquire or<br />
retain: ... Microbial or other biological agents, or toxins whatever<br />
their origin or method of production, of types and in wantities that<br />
have no justification for prophylactic, protective or other peaceful<br />
purpose..." ''<sup>3</sup></blockquote><br />
</li><br />
<li> Breach of the BTWC by the Soviet Union which continued their<br />
offensive biologicla warfare programm </li><br />
<li> Stop of the biological warfare Program "Biopreparat" accompanies<br />
the Dissolution of the Svoviet Union in 1991</li><br />
<li> Antrax attacks in the USA in 2001</li><br />
After this short description of misused biological components there is<br />
still the question what impact biological warfare will have in the<br />
future of mankind.<br><br />
<br><br />
</ul><br />
<center><br />
<h2>Broad avilibility of knowledge</h2><br />
</center><br />
n the life sciences information has allways been freely acessible for<br />
everybody who is interested in the results of a particular research<br />
project. The combination of this global availibility with the new<br />
possibilities of the internet broad for literature search and<br />
availibility of gene sequences made it easiere to collect informations<br />
for a possible misuse. <br><br />
For the overwhelming majority this open availibility is absolutely<br />
desirely but on the other hand there are also examples of research<br />
results that bear a very high risk to be misused. <br><br />
Following we present the three most controversial discussed<br />
publications that could also be read as a "How to create your own<br />
bioweapon".<br />
<h3>Mousepox Virus in Australien</h3><br />
<br><br />
The Australian research group around <a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/11152493"> Jackson et al. ;<br />
2001 <sup>16</sup></a>inserted the coding region of the IL-4 gne into<br />
the genome of mousepox. They hoped to create a virus that sterilizes<br />
mice and thus provides a means for pest control. Contrarely to their<br />
expectations they had created a superstrain that killed even naturally<br />
resistent mice and mice that had been vaccinated against normal<br />
mousepox. This discovery could potentially be used to make smallpox<br />
resistant to potential vaccines.<br />
<h3>Synthesis of a Polio Virus</h3><br />
<br><br />
At the State University of New York <a<br />
href="http://www.sciencemag.org/cgi/content/full/297/5583/1016"> Cello<br />
et al. ; 2002 <sup>05</sup> synthesized a "living" polio virus from<br />
scratch. Especially the </a><a<br />
href="http://www.sciencemag.org/cgi/content/full/1072266/DC1">Supporting<br />
Online Material</a> caused worried remarks because it precisely<br />
describes how to assemble a virus from small oligo nucleotides. The<br />
authors jusifyed their project by declaring that they:<br />
<blockquote>"made the virus to send a warning that terrorists might be<br />
able to make biological weapons without obtaining a natrual virus"<a<br />
href="http://www.ncbi.nlm.nih.gov/pubmed/19784453"><sup>17</sup></a></blockquote><br />
<h3>Reconstitution of the Spanish Flu</h3><br />
<br><br />
<img style="width: 300px; height: 223px;" class="right"<br />
src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Spanishflu.jpg"<br />
alt="Influenza hospital for american soldiers" align="left"><br />
At the Center for Disease Control and Prevention (CDC) the group around<br />
<a href="http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf">Tumpey<br />
et al. ; 2005<sup>24</sup></a> published that they had sequenced and<br />
recreated the pandemic Spanish Flu Virus of 1918 which killed 20-50<br />
million people. The publication of the sequence provoked several very<br />
different responses that can only be partially be quoted here.<br />
The controversial noticed outrider of the Synthetic Biology Craig<br />
Venter seen in the new virus<br />
<blockquote>"the first true Juressic Parc scenario" <a<br />
href="http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&amp;sID=4233"><sup>2</sup></a></blockquote><br />
The Institute Professor at the MIT Philip A. Sharp supported the<br />
publication because he:<br />
<blockquote>"belive[s] that allowing the publication of this<br />
information was the correct decision in terms of both national security<br />
and public health" <a<br />
href="http://www.sciencemag.org/cgi/content/short/310/5745/17"><sup>12</sup></a></blockquote><br />
Wheras <a<br />
href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf">v.<br />
Bubnoff; 2005<sup>25</sup></a> critisizes the CDS for it's careless<br />
regulations for the shipment of viruses and the willingness to<br />
propagate highly virulent viruses. This causes in his opinion the risk<br />
of possible accidents resulting in the release of the virus to the<br />
environment.<br><br />
<br><br />
<h3>Availability of molecular biological techniques</h3><br />
<br />
The knowledge required for the creation of a genetically engineered virus can easily be accessed from all over the world in online gene banks and publication databases and could be potentially be used for a destructive purpose <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819443"><sup>20</sup></a>. In 2007, Scott C. Mohr published the first part of his manuscript <a href="http://openwetware.org/images/3/3d/SB_Primer_100707.pdf">Primer for Synthetic Biology<sup>14</sup></a></li>. This open accessible document explaining the basics of molecular engineering addresses people interested in genetics but coming from a non-academic background. This development called "garage biology" or "biohacking" is a clear indication for the tendency to have knowledge about molecular biology available in society, as it was seen with programming and computer hacking from ~1980 on.<br />
<br />
<h3>Availability of synthesized DNA</h3><br />
<br />
<br />
One of the reactions to the publication of the genome sequence of the Spanish flu strain from 1918 was <a href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf"> v. Bubnoff, 2005<sup>25</sup></a></li> who pointed out the lax handling of the reconstituted virus sequence, the ease of availability and the low effort that has to be invested to recreate a virus:<br />
<blockquote>''"Scientists in Canada are planning to work with the virus, although they will not request it from the CDC. [...] Reconstructing the live virus from its DNA would then take just a few days, he says.''</blockquote><br />
Technical advances and dropping prices in the field of gene synthesis brings several deadly germs into the range of small research projects or even private persons as for example Spanish Flu (Influenza H<sub>1</sub>N<sub>1</sub>) with a 13.5 kbp <a href=http://expasy.org/viralzone/all_by_species/131.html><sup>*</sup></a> or even the Ebola virus with a 19 kbp genome <a href=http://expasy.org/viralzone/all_by_species/207.html><sup>*</sup></a>.<br />
<br />
In 2006, the “Guardian” journalist James Randson ordered a 78 bp DNA oligonucleotide coding for the capsid of the smallpox <i>Orthopoxvirus variola</i> with the intention to alert the public.<br />
<a href="http://www.guardian.co.uk/world/2006/jun/14/terrorism.topstories3"><sup>10</sup></a> <br><br />
In the United States, smallpox are listed as schedule 5 pathogens in the <i>Anti-terrorism, Crime and Security Act of 2001</i> and are illegal to keep or use without first notifying the civil authorities.<br><br />
On the other hand, it is questionable if these prohibitions or perhaps the size of the smallpox genome of ~200 kb <a href=http://expasy.org/viralzone/all_by_species/149.html><sup>*</sup></a> are sufficient to prevent de novo synthesis in the future efficiently. His intention was to focus the public's attention to the possibility that one of the most severe plagues in the history of mankind could be synthesized and released to the environment, either intentionally or by mistake. This would be a tragic setback since the smallpox's eradication was celebrated in 1979 by the World Health Organization as one of the greatest victories in the history of medicine <a href=http://www.who.int/mediacentre/factsheets/smallpox/en><sup>33</sup></a>.<br><br />
In order to prevent the unauthorized synthesis of gene sequences encoding hazardous biological agents, efforts to implement sequence analysis algorithms into the operating procedure of all organizations and companies capable of synthesizing gene sequences are being undertaken.<br> <br><br />
This barrier should be implemented not on a voluntary basis but as a legally binding regulation. It should be enforced by the government on a national level, but effort should also be made to find solutions for an international progress on this issue.<br><br />
As a first step, the development of search algorithms as Craic's BlackWatch<a<br />
href="https://biotech.craic.com/blackwatch/introduction.html><sup>34</sup></a> should be promoted and refined.<br><br><br />
Additional to this sequence base search for possible misuse of gene sequences, each order of already existing or synthesized genes could first be aligned with a list of countries, and in a second search with the so called Hadex exclusion list that names people and organizations excluded from obtaining dual-use gene material<a href=http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf><sup>1</sup></a>. <br />
<br><br />
<h2>The nature of biological weapons - Who is willing to accept their drawbacks</h2><br />
When considering the use of biological means for warfare, a potential aggressor has to accept several serious drawbacks. Biological weapons are not fully controllable, harmful effects to the civilian population cannot be foreseen. If replication-potent germs were used for such a purpose, they could possibly mutate and seriously harm mankind or other animal populations.<br><br />
This makes biological warfare unattractive for most nations, especially because more predictable weapons exist that can easily be directed against military targets with limited collateral damage.<br />
Unfortunately, national warfare programs are not the only source of danger when considering biological warfare. <br />
<blockquote>"An increasing number of countries believe that their political and security interests could be protected or achieved only through the possession of such weapons, especially in view of the overwhelming superiority of the US armed forces in terms of conventional weapons."<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a></blockquote> <br />
Several countries around the globe are technically capable to develop biological weapons or to support terroristic groups in doing so. When this physical capability meets an ideological attitude that is based on contempt for other concepts of society, it cannot be ruled out that biological warfare is taken into consideration. <br />
<br />
<h2>Synthetic Biology vs. Nuclear Research - Consideration in terms of security policy<h3> <br />
When considering the history of research, the dual-use character of technical innovations has always been present, only the reaction of society to it differs from case to case. As an example, scientific discoveries in the field of nuclear physics where treated as classified information having a high relevance for the national security as shown in the case of the Manhattan project<a href="http://www.ncbi.nlm.nih.gov/pubmed/19784453"><sup>17</sup></a>. A comparable censorship is not established in life sciences research apart of some cases when national security was endangered.<br><br><br />
Another point concerning the threat arising from biological weapons is the absence of methods that can be used to monitor such weapons of mass destruction as pointed out by M.R. Dando. This fact and the increasing world-wide mobility would make it impossible to prevent the spreading of such weapons when once accessible to potential assassins. <br />
<br />
<h3>Is a revision of basic research necessary in sensitive research fields? </h3><br />
The publication of research papers containing dual-use knowledge caused several people to call for regulation. In the United States, the so called Fink Committee evaluated the possibility that research in life sciences could be used for biological warfare purposes and how this could be avoided.<a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br><br><br />
<br />
This committee had two recommendations:<br />
<ul><br />
<li>To familiarize the scientific community with the dual-use problem in the life sciences.</li><br />
<li>To review experiments of concern. Experiments are of concern if they:<br />
<ul><br />
<li>render a vaccine ineffective</li><br />
<li>confer resistance to therapeutically useful antibiotics or<br />
antiviral agents</li><br />
<li>enhance the virulence of a pathogen or render a nonpathogen<br />
virulent</li><br />
<li>increase transmissibility of a pathogen</li><br />
<li>alter the host range of a pathogen</li><br />
<li>enable the evasion of diagnostic/detection tools</li><br />
<li>enable the weaponization of a biological agent or toxin</li></ul><br />
<li>To review publications with a strong dual-use factor</li><br />
<li>To create a National Science Advisory Board</li><br />
<li>To improve the oversight and reduce unauthorized accessibility of hazardous gene material</li><br />
<li>To include the life sciences into the efforts for national security</li><br />
<li>To harmonize the international oversight over dual-use research</li><br />
</ul><br />
<br />
The implementation of these recommendations would be desirable, even though they might cause inconveniences for scientists working in the affected fields.<br />
<br />
<br />
<br />
<br />
<h2>Conclusion</h2><br />
<br />
<br />
Concluding all factors mentioned above, we need to consider the possibility that a person, organization or state could misuse the fast-advancing life sciences for biological warfare. The possibilities and the simplicity of dual-use research misuse will become more and easier the faster the scientific progress advances.<br><br />
Therefore it is important to minimize these potential risks before they become reality. This is not only the task of a designated group but a moral obligation for scientists, politicians and everybody related to dual-use research. Especially scientists have to contribute to and lead the continuing discussion on this topic because they are able to estimate how aspects of their research fields might be misused.<br />
<br />
<center><br />
<h2>Conclusion</h2><br />
<br><br />
<table style="text-align: left; width: 90%;" border="0" cellpadding="2"<br />
cellspacing="2"><br />
<tbody><br />
<tr><br />
<td style="vertical-align: top;"><img<br />
src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150"></td><br />
<td style="vertical-align: middle;"><span<br />
style="color: DarkGreen;"><b>Include<br />
something in your project<br />
description and presentations that demonstrates that you have thought<br />
about how others could misuse your work.</b></span> </td><br />
</tr><br />
</tbody><br />
</table><br />
<br><br />
</center><br />
<p>For sure there is allway the possibility that knowledge to produce<br />
transgene viral vectors could be used to produce bioweapons. Therefor<br />
it was important for us to use a system that does not bear the risk<br />
that someone could use it for evil purpose. In the case of the<br />
Adeno-associated virus the very limited packaging capacity is the major<br />
reason that excludes it from the list of agents that could<br />
realistically be used for the pruduction of bioweapons. Even a fully<br />
replication potent AAV will depend on the coninfection of a helpervirus<br />
and is therefore not suitable for a fast propagation in an population.<br />
Additional to this point we concentrated our project on the retargeting<br />
of the virus - means to make the broad tropismn more narrow and to<br />
decrease the transduction efficiency in the most cases. This<br />
modification is usually mainly required for medical purposes. Also we<br />
did neither investigate possibilities to shield the vector from the<br />
immune system of potential host nor ways to bypass an existing<br />
immunity. </p><br />
<br><br />
<br><br />
<ul><br />
<li>01 <a<br />
href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf">Synthetic<br />
Biology &amp; Biosecurity - Awareness in Europe | Kelle ; 2007<sup>1</sup></a></li><br />
<li>02 <a<br />
href="http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&amp;sID=4233">Synthetic<br />
Biology - Social and Ethical Challenges | Balmer et Martin ; 2008<sup>2</sup></a></li><br />
<li>03 Synthetic Biology - Applying Engineering to Biology <br><br />
used </li><br />
<li>04 <a<br />
href="http://www.informaworld.com/smpp/content%7Econtent=a713604665&amp;db=all">The<br />
Impact of the Development of Modern Biology and Medicine on the<br />
Evolution of Offensive Biological Warfare Programs in the Twentieth<br />
Century | Dando ; 1999 <sup>04</sup></a></li><br />
<li>05 <a<br />
href="http://www.sciencemag.org/cgi/content/full/297/5583/1016">Chemical<br />
Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the<br />
Absence of Natural Template | Cello et al. ; 2002 <sup>05</sup></a></li><br />
<li><a href="http://www.sciencemag.org/cgi/content/full/297/5583/1016">06<br />
</a><a<br />
href="http://www.nytimes.com/2003/12/03/opinion/03iht-edstein_ed3_.html">Dangerous<br />
research : When science breeds nightmares | Steinbruner et Harris ;<br />
2003 <sup>06</sup></a></li><br />
<li>07 <a<br />
href="http://www.jcvi.org/cms/fileadmin/site/research/projects/synthetic-genomics-report/synthetic-genomics-report.pdf">Synthetic<br />
Genomics - Options for governance | Garfinkel et al. ; 2007 <sup>07</sup></a></li><br />
<li>08 <a<br />
href="http://www.nature.com/nrg/journal/v6/n7/abs/nrg1637.html">Synthetic<br />
biology | Benner et Sismour , 2005 <sup>08</sup></a></li><br />
<li>09 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16954140">Synthetic<br />
biology—putting engineering into biology | Heinemann et Panke ; 2006 <sup>09</sup></a></li><br />
<li>10 <a<br />
href="http://www.guardian.co.uk/world/2006/jun/14/terrorism.topstories3">Revealed:<br />
the lax laws that could allow assembly of deadly virus DNA | Randerson<br />
; 2006 <sup>10</sup></a> </li><br />
<li>11 <a<br />
href="http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf">The<br />
Promise and Perils of Synthetic Biology | Tucker &amp; Zilinskas ; 2006<br />
<sup>11</sup></a></li><br />
<li>12 <a<br />
href="http://www.sciencemag.org/cgi/content/short/310/5745/17">1918<br />
Flu and Responsible Science | Sharp ; 2005 <sup>12</sup></a></li><br />
<li>13 <a href="http://www.fas.org/irp/cia/product/bw1103.pdf"> The<br />
Darker Bioweapons Future | CIA ; 2003 <sup>13</sup></a></li><br />
<li>14 <a href="http://openwetware.org/images/3/3d/SB_Primer_100707.pdf">Primer for Synthetic Biology | Mohr ; 2007 <sup>14</sup></a></li><br />
<li>15 [[Media:Freiburg10_The bugs of war.pdf]] </li><br />
<li>16 <a href="http://www.ncbi.nlm.nih.gov/pubmed/11152493">Expression<br />
of mouse interleukin-4 by a recombinant ectromelia virus suppresses<br />
cytolytic lymphocyte responses and overcomes genetic resistance to<br />
mousepox. | Jackson et al. ; 2001 <sup>16</sup></a></li><br />
<li>17 <a href="http://www.ncbi.nlm.nih.gov/pubmed/19784453">Governance<br />
of dual-use research: an ethical dilemma. | Selgelid ; 2009 <sup>17</sup></a></li><br />
<li>18 <a href="http://www.ncbi.nlm.nih.gov/pubmed/18625678">Evidence-based<br />
biosafety: a review of the principles and effectiveness of<br />
microbiological containment measures. | Kimman et al. 2008 <sup>18</sup></a><br />
</li><br />
<li>19 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819452">A<br />
Hippocratic Oath for life scientists | Revill et Dando ; 2006 <sup>19</sup></a></li><br />
<li>20 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819443">Empowerment<br />
and restraint in scientific communication. New developments make it<br />
easier to share information, but more difficult to deal with dual-use<br />
biology. | Campbell ; 2006<sup>20</sup></a></li><br />
<li>21 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16819441">When<br />
risk<br />
outweighs benefit | Aken ; 2006 <sup>21</sup></a></li><br />
<li>22 <a href="http://www.ncbi.nlm.nih.gov/pubmed/12789408">Advances<br />
in life sciences and bioterrorism. Risks, perspectives and<br />
responsibilities.| Beck ; 2003 <sup>22</sup></a></li><br />
<li>23 <a href="http://www.ncbi.nlm.nih.gov/pubmed/12590130">PNAS<br />
policy on publication of sensitive material in the life sciences |<br />
Cozzarelli ; 2003 <sup>23</sup></a></li><br />
<li>24 <a<br />
href="http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf">Characterization<br />
of the<br />
Reconstructed 1918 Spanish<br />
Influenza Pandemic Virus | Tumpey et al. ; 2005<sup>24</sup></a> </li><br />
<li>25 <a<br />
href="http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf">Deadly<br />
flu virus can be<br />
sent through the mail| v. Bubnoff; 2005<sup>25</sup></a></li><br />
<li>26 <a<br />
href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf">Risk<br />
assessment of human Adeno-associated viruses| ZKBS; 2001<sup>26</sup></a></li><br />
<li>27 <a<br />
href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf">Advises<br />
for AAV carrying cell cycle regulating genes| ZKBS; 2004 <sup>27</sup></a></li><br />
<li>28 <a<br />
href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf">Risk<br />
assessment of human Adeno-associated viruses and AAV derived vectors|<br />
ZKBS; 2005 <sup>28</sup></a> </li><br />
<li>29 <a href="http://www.ncbi.nlm.nih.gov/pubmed/10700178">Evidence<br />
for gene transfer and expression of factor IX in haemophilia B patients<br />
treated with an AAV vector.| Kai et al. ; 2000 <sup>29</sup></a><br />
Bioverteilung in Klinischer Studie </li><br />
<li>30 <a<br />
href="https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf">Biosafety<br />
application of the iGEM team Freiburg_Bioware 2010<sup>30</sup></a> (in<br />
German)</li><br />
<li>31 <a<br />
href="https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg">Official<br />
classification as Biological Safety Level 1 by the local biosafety<br />
office<sup>31</sup></a></li><br />
<li>32 <a<br />
href="http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm">Appendix<br />
B | National Institute of Health <sup>32</sup></a></li><br />
<li>33 <a href="http://www.who.int/mediacentre/factsheets/smallpox/en"> <br />
Media centre - Smallpox | World Health Organisation <sup>33</sup></a></li><br />
<li>34 <a<br />
href="https://biotech.craic.com/blackwatch/introduction.html>BlackWatch Homepage | Craic computing<sup>34</sup></a></li><br />
<br />
<br />
<li>34 <a<br />
href="https://biotech.craic.com/blackwatch/introduction.html>BlackWatch Homepage | Craic computing<sup>34</sup></a></li><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</ul><br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-10-28T01:49:59Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<div class="virus"><img src="https://static.igem.org/mediawiki/2010/2/24/Freiburg10_rot250trans.gif" title="virus (3.5MB)" id="animated_virus"/></div><br />
<div class="div_home"><br />
<p><br />
Gene delivery using viral vectors holds great promise for the treatment of acquired and inherited diseases. The human Adeno-Associated Virus (AAV) is a small, non-pathogenic, single-stranded DNA virus gaining increasing attention being both versatile and effective. Taking current knowledge into account, we generated a recombinant, modularized, BioBrick-compatible AAV ‘Virus Construction Kit’. We provide parts for modified capsid proteins, targeting modules, tumor-specific promoters, and prodrug-activating enzymes as well as readily assembled vectors for gene delivery and production of non-replicative virus particles. The viral tropism is altered by N-terminal fusion or by loop replacement of the capsid proteins. Functionality of viruses constructed from our kit was demonstrated by fluorescent protein expression in infected cells and by prodrug-induced killing of tumor cells upon viral delivery of a thymidine kinase. Incorporating multiple layers of safety, we provide a general tool to the growing field of personalized medicine and demonstrate its use in tumor therapy.<br />
</p><br />
</div><br />
<br />
<!---Box on the upper left: Project Results---><br />
<div class="box_home"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project"><img class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><br />
</a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results"><h2>Project Results</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><h5>Highlights: </h5></a>--><br />
Customized therapeutic AAV Vectors!<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#modularization"><span class="bold">[more]</span></a></p><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><span class="bold"><h5>Heading:</h5></span></a>--><br />
Differential Tumor Targeting!<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#targeting"><span class="bold">[more]</span></a></p><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold"><h5>Head-Line:</h5></span></a>--><br />
Prodrug-activated tumor cell killing!<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#arming"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!---Big box on the left: Virus Construction Kit - Manual---><br />
<br />
<div class="box_long"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_Manual_Logo_small.png" id="ccc" /><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h2>Virus Construction Kit - The Manual</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h5>Heading:</h5></a>--><br />
Everything you need to know about AAV vectors <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!--- News Ticker ---><br />
<br />
<div class="box_long box_long_news news_ticker"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h2>Statistics - In a Minute</h2></a><br />
<ul id="news" class="newsticker"><br />
<li><div class="news"><img class="right" src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_Number_of_Plasmids.png" id="ccc" /><p><br />
What happened in the lab during iGEM? </p><br />
</div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_Longest_Wikipage.png"<br />
alt="Longest Wikipage" width="368"><p>Longest Wikipage:<br />with 222,795 bytes</p></div><br />
</li><br />
<li><div class="news"><img src="https://static.igem.org/mediawiki/2010/1/10/Freiburg10_Number_of_Biobricks.png"<br />
alt="Number of BioBricks" width="368"><p>Number of Biobricks:<br /> 116</p></div></li><br />
<li><div class="news"><img heigth="100px" src="https://static.igem.org/mediawiki/2010/3/3f/Freiburg10_Number_of_ordered_Oligo-Nucleotides.png"<br />
alt="Number of ordered Oligo-Nucleotides" width="184"><p>Number of Ordered Oligos:<br /> 193</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f3/Freiburg10_Minipreps_total.png"<br />
alt="Minipreps total" width="184"><p>Mini-Preps total:<br /> 1085</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f7/Freiburg10_Midipreps_total.png"<br />
alt="Midipreps total" width="368"><p>Midi-Preps total:<br /> 106</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d6/Freiburg10_Highest_Prep_concentration.png"<br />
alt="Highest Prep concentration (Midi)" width="368"><p>Highest Midi-Prep Concentration:<br />5045,6 ng/µL </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/af/Freiburg10_Number_of_Glyzerolstocks.png"<br />
alt="Number of Glycerolstocks" width="368"><p>Number of Glycerolstocks:<br /> 763 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/6/60/Freiburg10_Number_of_Team_members.png"<br />
alt="Number of Team members" width="368"><p>Number of Team members:<br /> 15 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/0/03/Freiburg10_Days_in_the_lab.png"<br />
alt="Days in the lab" width="368"><p>Days in the lab:<br /> 170 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_Longest_Labday.png"<br />
alt="Longest Labday" width="368"><p>Longest Labday:<br />18h </p></div></</li><br />
<li><div class="news"><img height="100px"<br />
src="https://static.igem.org/mediawiki/2010/b/b5/Freiburg10_Coffee_consumed_during_iGEM.png"<br />
alt="Coffee consumed during iGEM" width="184"><p>Coffee Consumed During iGEM:<br /> 2238 Cups</p></div></li><br />
<li><div class="news"><img heigth="100px"class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><p><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h5>!Freiburg Bioware Statistics!</h5></a></p></div></li><br />
</ul><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<div height="290px"><br />
<!---Box on the upper right: BioBricks---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><img class="right" src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg_10_BioBrick_icon_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><h2>BioBricks</h2></a><br />
<p class="standard">Modularization and modification of the viral capsids for retargeting approaches and directed gene delivery of suicide genes resulted in many different BioBricks.<br> Click here to explore them.</p><br />
</div><br />
<br />
<!---Box on the lower left: Biosafety---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><img class="right" src="https://static.igem.org/mediawiki/2010/f/f9/Freiburg10_Danger_Virus_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><h2>Biosafety</h2></a><br />
<p class="standard">The aspect of biological safety was considered well and a risk assessment and security profile for the Adeno-associated Virus were created. Read more about Biosafety issues..</p><br />
<br />
</div><br />
<br />
<!---Box on the lower right: Notebook---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/95/Freiburg_10_Notebook_image_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<h2>Notebook</h2></a><br />
<p><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><span class="bold"><h5>Only cloning and cell culture? </h5></span></a><br /><br />
No! Besides <br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal"><span class="bold">BioBrick assembly</span></a>, flow cytometry analysis, quantification based methos via ELISA or Western Blot and cytotoxicity assays have been performed <a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Methods"><span class="bold"><h5>(go to Methods)</h5></span></p><br />
</a><br />
</div><br />
<br />
<br />
<!---Box on the lower right: Team---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_Virus_Logo_Small_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<h2>Team</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><h5>Fun and Science at the same time?</h5></a></a><br /><br />
Yes! The iGEM team Freiburg did not only share the lab bench, but as well some nice days <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Photo_Gallery"><span class="bold">canoeing</span></a> and initiated the photo contest <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Cuckoo_Clock"><span class="bold">Cuckoo Clock Competition</span></a></p><br />
</div><br />
</div><br />
<div class="clearfix"></div><br />
<br /><br />
<h1>Sponsors</h1><br />
<br />
<div class="map" height="1169px"><br />
<map name="Sponsoren_iGEM" id="Sponsoren_iGEM"><br />
<area shape="circle" coords="705,369,65" href="http://www.uni-potsdam.de/" target="_blank" alt="Uni Potsdam" title="Uni Potsdam" /><br />
<area shape="circle" coords="156,138,121" href="http://www.uni-freiburg.de/start-en.html?set_language=en" target="_blank" alt="Uni Freiburg" title="Uni Freiburg" /><br />
<area shape="rect" coords="304,8,815,269" href="http://www.bioss.uni-freiburg.de/cms/index.php#" target="_blank" alt="Bioss" title="Bioss" /><br />
<area shape="rect" coords="501,311,611,434" href="http://www.molbiotech.uni-freiburg.de/" target="_blank" alt="kuk.lab" title="kuk.lab" /><br />
<area shape="rect" coords="274,329,480,412" href="http://www.daad.de/en/index.html" alt="daad" target="_blank" title="daad" /><br />
<area shape="rect" coords="67,324,238,419" href="http://www.frias.uni-freiburg.de/" alt="frias" target="_blank" title="frias" /><br />
<area shape="rect" coords="95,534,376,686" href="http://www.roche.com/index.htm" target="_blank" alt="Roche" title="Roche" /><br />
<area shape="rect" coords="510,458,677,606" href="http://www.qiagen.com/default_qs.aspx" target="_blank" alt="Qiagen" title="Qiagen" /><br />
<area shape="rect" coords="477,671,708,752" href="http://www.starlab.de/int/?l=2" target="_blank" alt="Starlab" title="Starlab" /><br />
<area shape="rect" coords="551,799,787,861" href="http://www.peqlab.com" target="_blank" alt="peqlab" title="peqlab" /><br />
<area shape="rect" coords="291,801,494,859" href="http://www.eppendorf.com/int/?l=1&amp;action=start" target="_blank" alt="Eppendorf" title="Eppendorf" /><br />
<area shape="rect" coords="43,801,226,859" href="http://www.gilson.com/en/" target="_blank" alt="Gilson" title="Gilson" /><br />
<area shape="rect" coords="44,872,218,936" href="http://www.fermentas.de/index.php?&amp;language=en" target="_blank" alt="Fermentas" title="Fermentas" /><br />
<area shape="rect" coords="42,942,259,1002" href="http://www.home.agilent.com" target="_blank" alt="aigilent" title="aigilent" /><br />
<area shape="rect" coords="289,958,388,1041" href="http://www.avidity.com/" target="_blank" alt="Avidity" title="Avidity" /><br />
<area shape="rect" coords="412,960,566,1048" href="http://www.hiss-dx.de/hiss/index.php?id=43&amp;L=1" target="_blank" alt="Hiss" title="Hiss" /><br />
<area shape="rect" coords="591,981,795,1043" href="http://www.biozym.com/site/21/Produkte.aspx" target="_blank" alt="Biozym" title="Biozym" /><br />
<area shape="rect" coords="592,876,750,956" href="http://www.gatc-biotech.com" target="_blank" alt="GATC" title="GATC" /><br />
<area shape="rect" coords="296,886,540,948" href="http://www.geneart.com/" target="_blank" alt="Geneart" title="Geneart" /><br />
<area shape="rect" coords="495,1070,808,1151" href="http://www.atg-biosynthetics.com/" target="_blank" alt="ATG" title="ATG" /><br />
<area shape="rect" coords="242,1086,469,1154" href="http://www.purimex.com/" alt="purimex" target="_blank" title="purimex" /><br />
<area shape="rect" coords="45,1084,219,1154" href="http://www.dkfz.de/index.html" alt="dkfz" target="_blank" title="dkfz" /><br />
<area shape="rect" coords="46,1021,264,1075" href="http://www.mathworks.com" target="_blank" alt="Mathworks" title="Mathworks" /><br />
</map><br />
<img src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Sponsoren_Image_Map.gif" width="826" height="1169" border="0" alt="" title="" usemap="#Sponsoren_iGEM" /><br />
</div><br />
<br />
<br /><br />
<br /><br />
<center><a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
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<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-10-28T01:42:59Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<div class="virus"><img src="https://static.igem.org/mediawiki/2010/2/24/Freiburg10_rot250trans.gif" title="virus (3.5MB)" id="animated_virus"/></div><br />
<div class="div_home"><br />
<p><br />
Gene delivery using viral vectors holds great promise for the treatment of acquired and inherited diseases. The human Adeno-Associated Virus (AAV) is a small, non-pathogenic, single-stranded DNA virus gaining increasing attention being both versatile and effective. Taking current knowledge into account, we generated a recombinant, modularized, BioBrick-compatible AAV ‘Virus Construction Kit’. We provide parts for modified capsid proteins, targeting modules, tumor-specific promoters, and prodrug-activating enzymes as well as readily assembled vectors for gene delivery and production of non-replicative virus particles. The viral tropism is altered by N-terminal fusion or by loop replacement of the capsid proteins. Functionality of viruses constructed from our kit was demonstrated by fluorescent protein expression in infected cells and by prodrug-induced killing of tumor cells upon viral delivery of a thymidine kinase. Incorporating multiple layers of safety, we provide a general tool to the growing field of personalized medicine and demonstrate its use in tumor therapy.<br />
</p><br />
</div><br />
<br />
<!---Box on the upper left: Project Results---><br />
<div class="box_home"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project"><img class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><br />
</a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results"><h2>Project Results</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><h5>Highlights: </h5></a>--><br />
Customized therapeutic AAV Vectors!<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#Modularization"><span class="bold">[more]</span></a></p><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><span class="bold"><h5>Heading:</h5></span></a>--><br />
Differential Tumor Targeting!<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#Targeting"><span class="bold">[more]</span></a></p><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold"><h5>Head-Line:</h5></span></a>--><br />
Prodrug-activated tumor cell killing!<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#Arming"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!---Big box on the left: Virus Construction Kit - Manual---><br />
<br />
<div class="box_long"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_Manual_Logo_small.png" id="ccc" /><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h2>Virus Construction Kit - The Manual</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h5>Heading:</h5></a>--><br />
Everything you need to know about AAV vectors <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!--- News Ticker ---><br />
<br />
<div class="box_long box_long_news news_ticker"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h2>Statistics - In a Minute</h2></a><br />
<ul id="news" class="newsticker"><br />
<li><div class="news"><img class="right" src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_Number_of_Plasmids.png" id="ccc" /><p><br />
What happened in the lab during iGEM? </p><br />
</div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_Longest_Wikipage.png"<br />
alt="Longest Wikipage" width="368"><p>Longest Wikipage:<br />with 222,795 bytes</p></div><br />
</li><br />
<li><div class="news"><img src="https://static.igem.org/mediawiki/2010/1/10/Freiburg10_Number_of_Biobricks.png"<br />
alt="Number of BioBricks" width="368"><p>Number of Biobricks:<br /> 116</p></div></li><br />
<li><div class="news"><img heigth="100px" src="https://static.igem.org/mediawiki/2010/3/3f/Freiburg10_Number_of_ordered_Oligo-Nucleotides.png"<br />
alt="Number of ordered Oligo-Nucleotides" width="184"><p>Number of Ordered Oligos:<br /> 193</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f3/Freiburg10_Minipreps_total.png"<br />
alt="Minipreps total" width="184"><p>Mini-Preps total:<br /> 1085</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f7/Freiburg10_Midipreps_total.png"<br />
alt="Midipreps total" width="368"><p>Midi-Preps total:<br /> 106</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d6/Freiburg10_Highest_Prep_concentration.png"<br />
alt="Highest Prep concentration (Midi)" width="368"><p>Highest Midi-Prep Concentration:<br />5045,6 ng/µL </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/af/Freiburg10_Number_of_Glyzerolstocks.png"<br />
alt="Number of Glycerolstocks" width="368"><p>Number of Glycerolstocks:<br /> 763 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/6/60/Freiburg10_Number_of_Team_members.png"<br />
alt="Number of Team members" width="368"><p>Number of Team members:<br /> 15 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/0/03/Freiburg10_Days_in_the_lab.png"<br />
alt="Days in the lab" width="368"><p>Days in the lab:<br /> 170 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_Longest_Labday.png"<br />
alt="Longest Labday" width="368"><p>Longest Labday:<br />18h </p></div></</li><br />
<li><div class="news"><img height="100px"<br />
src="https://static.igem.org/mediawiki/2010/b/b5/Freiburg10_Coffee_consumed_during_iGEM.png"<br />
alt="Coffee consumed during iGEM" width="184"><p>Coffee Consumed During iGEM:<br /> 2238 Cups</p></div></li><br />
<li><div class="news"><img heigth="100px"class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><p><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h5>!Freiburg Bioware Statistics!</h5></a></p></div></li><br />
</ul><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<div height="290px"><br />
<!---Box on the upper right: BioBricks---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><img class="right" src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg_10_BioBrick_icon_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><h2>BioBricks</h2></a><br />
<p class="standard">Modularization and modification of the viral capsids for retargeting approaches and directed gene delivery of suicide genes resulted in many different BioBricks.<br> Click here to explore them.</p><br />
</div><br />
<br />
<!---Box on the lower left: Biosafety---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><img class="right" src="https://static.igem.org/mediawiki/2010/f/f9/Freiburg10_Danger_Virus_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><h2>Biosafety</h2></a><br />
<p class="standard">The aspect of biological safety was considered well and a risk assessment and security profile for the Adeno-associated Virus were created. Read more about Biosafety issues..</p><br />
<br />
</div><br />
<br />
<!---Box on the lower right: Notebook---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/95/Freiburg_10_Notebook_image_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<h2>Notebook</h2></a><br />
<p><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><span class="bold"><h5>Only cloning and cell culture? </h5></span></a><br /><br />
No! Besides <br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal"><span class="bold">BioBrick assembly</span></a>, flow cytometry analysis, quantification based methos via ELISA or Western Blot and cytotoxicity assays have been performed <a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Methods"><span class="bold"><h5>(go to Methods)</h5></span></p><br />
</a><br />
</div><br />
<br />
<br />
<!---Box on the lower right: Team---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_Virus_Logo_Small_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<h2>Team</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><h5>Fun and Science at the same time?</h5></a></a><br /><br />
Yes! The iGEM team Freiburg did not only share the lab bench, but as well some nice days <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Photo_Gallery"><span class="bold">canoeing</span></a> and initiated the photo contest <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Cuckoo_Clock"><span class="bold">Cuckoo Clock Competition</span></a></p><br />
</div><br />
</div><br />
<div class="clearfix"></div><br />
<br /><br />
<h1>Sponsors</h1><br />
<br />
<div class="map" height="1169px"><br />
<map name="Sponsoren_iGEM" id="Sponsoren_iGEM"><br />
<area shape="circle" coords="705,369,65" href="http://www.uni-potsdam.de/" target="_blank" alt="Uni Potsdam" title="Uni Potsdam" /><br />
<area shape="circle" coords="156,138,121" href="http://www.uni-freiburg.de/start-en.html?set_language=en" target="_blank" alt="Uni Freiburg" title="Uni Freiburg" /><br />
<area shape="rect" coords="304,8,815,269" href="http://www.bioss.uni-freiburg.de/cms/index.php#" target="_blank" alt="Bioss" title="Bioss" /><br />
<area shape="rect" coords="501,311,611,434" href="http://www.molbiotech.uni-freiburg.de/" target="_blank" alt="kuk.lab" title="kuk.lab" /><br />
<area shape="rect" coords="274,329,480,412" href="http://www.daad.de/en/index.html" alt="daad" target="_blank" title="daad" /><br />
<area shape="rect" coords="67,324,238,419" href="http://www.frias.uni-freiburg.de/" alt="frias" target="_blank" title="frias" /><br />
<area shape="rect" coords="95,534,376,686" href="http://www.roche.com/index.htm" target="_blank" alt="Roche" title="Roche" /><br />
<area shape="rect" coords="510,458,677,606" href="http://www.qiagen.com/default_qs.aspx" target="_blank" alt="Qiagen" title="Qiagen" /><br />
<area shape="rect" coords="477,671,708,752" href="http://www.starlab.de/int/?l=2" target="_blank" alt="Starlab" title="Starlab" /><br />
<area shape="rect" coords="551,799,787,861" href="http://www.peqlab.com" target="_blank" alt="peqlab" title="peqlab" /><br />
<area shape="rect" coords="291,801,494,859" href="http://www.eppendorf.com/int/?l=1&amp;action=start" target="_blank" alt="Eppendorf" title="Eppendorf" /><br />
<area shape="rect" coords="43,801,226,859" href="http://www.gilson.com/en/" target="_blank" alt="Gilson" title="Gilson" /><br />
<area shape="rect" coords="44,872,218,936" href="http://www.fermentas.de/index.php?&amp;language=en" target="_blank" alt="Fermentas" title="Fermentas" /><br />
<area shape="rect" coords="42,942,259,1002" href="http://www.home.agilent.com" target="_blank" alt="aigilent" title="aigilent" /><br />
<area shape="rect" coords="289,958,388,1041" href="http://www.avidity.com/" target="_blank" alt="Avidity" title="Avidity" /><br />
<area shape="rect" coords="412,960,566,1048" href="http://www.hiss-dx.de/hiss/index.php?id=43&amp;L=1" target="_blank" alt="Hiss" title="Hiss" /><br />
<area shape="rect" coords="591,981,795,1043" href="http://www.biozym.com/site/21/Produkte.aspx" target="_blank" alt="Biozym" title="Biozym" /><br />
<area shape="rect" coords="592,876,750,956" href="http://www.gatc-biotech.com" target="_blank" alt="GATC" title="GATC" /><br />
<area shape="rect" coords="296,886,540,948" href="http://www.geneart.com/" target="_blank" alt="Geneart" title="Geneart" /><br />
<area shape="rect" coords="495,1070,808,1151" href="http://www.atg-biosynthetics.com/" target="_blank" alt="ATG" title="ATG" /><br />
<area shape="rect" coords="242,1086,469,1154" href="http://www.purimex.com/" alt="purimex" target="_blank" title="purimex" /><br />
<area shape="rect" coords="45,1084,219,1154" href="http://www.dkfz.de/index.html" alt="dkfz" target="_blank" title="dkfz" /><br />
<area shape="rect" coords="46,1021,264,1075" href="http://www.mathworks.com" target="_blank" alt="Mathworks" title="Mathworks" /><br />
</map><br />
<img src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Sponsoren_Image_Map.gif" width="826" height="1169" border="0" alt="" title="" usemap="#Sponsoren_iGEM" /><br />
</div><br />
<br />
<br /><br />
<br /><br />
<center><a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
</a></center><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-10-28T01:15:08Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<div class="virus"><img src="https://static.igem.org/mediawiki/2010/2/24/Freiburg10_rot250trans.gif" title="virus (3.5MB)" id="animated_virus"/></div><br />
<div class="div_home"><br />
<p><br />
Gene delivery using viral vectors holds great promise for the treatment of acquired and inherited diseases. The human Adeno-Associated Virus (AAV) is a small, non-pathogenic, single-stranded DNA virus gaining increasing attention being both versatile and effective. Taking current knowledge into account, we generated a recombinant, modularized, BioBrick-compatible AAV ‘Virus Construction Kit’. We provide parts for modified capsid proteins, targeting modules, tumor-specific promoters, and prodrug-activating enzymes as well as readily assembled vectors for gene delivery and production of non-replicative virus particles. The viral tropism is altered by N-terminal fusion or by loop replacement of the capsid proteins. Functionality of viruses constructed from our kit was demonstrated by fluorescent protein expression in infected cells and by prodrug-induced killing of tumor cells upon viral delivery of a thymidine kinase. Incorporating multiple layers of safety, we provide a general tool to the growing field of personalized medicine and demonstrate its use in tumor therapy.<br />
</p><br />
</div><br />
<br />
<!---Box on the upper left: Project Results---><br />
<div class="box_home"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project"><img class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><br />
</a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results"><h2>Project Results</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><h5>Highlights: </h5></a><br />
This Site is in Construction, later you will find more right here...<a href="https://2010.igem.org/Team:Freiburg_Bioware/Virus_Construction_Kit"><span class="bold">[more]</span></a></p><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Virus_Construction_Kit"><span class="bold"><h5>Heading:</h5></span></a><br />
This Site is in Construction, later on you will find more right here...<a href="https://2010.igem.org/Team:Freiburg_Bioware/Virus_Construction_Kit"><span class="bold">[Klick for more]</span></a></p><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold"><h5>Head-Line:</h5></span></a><br />
This Site is in Construction, later you will find more right here...<a href="https://2010.igem.org/Team:Freiburg_Bioware/Virus_Construction_Kit"><span class="bold">[Klick for more]</span></a></p><br />
</div><br />
<br />
<!---Big box on the left: Virus Construction Kit - Manual---><br />
<br />
<div class="box_long"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_Manual_Logo_small.png" id="ccc" /><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h2>Virus Construction Kit - The Manual</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h5>Heading:</h5></a>--><br />
Everything you need to know about AAV vectors! <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!--- News Ticker ---><br />
<br />
<div class="box_long box_long_news news_ticker"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h2>Statistics - In a Minute</h2></a><br />
<ul id="news" class="newsticker"><br />
<li><div class="news"><img class="right" src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_Number_of_Plasmids.png" id="ccc" /><p style="font-size: 12px;"><br />
What happened in the labduring iGEM? </p><br />
</div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_Longest_Wikipage.png"<br />
alt="Longest Wikipage" width="368"><p style="font-size: 12px;">Longest Wikipage:<br />with 222,795 bytes</p></div><br />
</li><br />
<li><div class="news"><img src="https://static.igem.org/mediawiki/2010/1/10/Freiburg10_Number_of_Biobricks.png"<br />
alt="Number of BioBricks" width="368"><p style="font-size: 12px;">Number of Biobricks:<br /> 116</p></div></li><br />
<li><div class="news"><img heigth="100px" src="https://static.igem.org/mediawiki/2010/3/3f/Freiburg10_Number_of_ordered_Oligo-Nucleotides.png"<br />
alt="Number of ordered Oligo-Nucleotides" width="184"><p style="font-size: 12px;">Number of Ordered Oligos:<br /> 193</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f3/Freiburg10_Minipreps_total.png"<br />
alt="Minipreps total" width="184"><p style="font-size: 12px;">Mini-Preps total:<br /> 1085</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f7/Freiburg10_Midipreps_total.png"<br />
alt="Midipreps total" width="368"><p style="font-size: 12px;">Midi-Preps total:<br /> 106</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d6/Freiburg10_Highest_Prep_concentration.png"<br />
alt="Highest Prep concentration (Midi)" width="368"><p style="font-size: 12px;">Highest Midi-Prep Concentration:<br />5045,6 ng/µL </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/af/Freiburg10_Number_of_Glyzerolstocks.png"<br />
alt="Number of Glycerolstocks" width="368"><p style="font-size: 12px;">Number of Glycerolstocks:<br /> 763 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/6/60/Freiburg10_Number_of_Team_members.png"<br />
alt="Number of Team members" width="368"><p style="font-size: 12px;">Number of Team members:<br /> 15 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/0/03/Freiburg10_Days_in_the_lab.png"<br />
alt="Days in the lab" width="368"><p style="font-size: 12px;">Days in the lab:<br /> 170 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_Longest_Labday.png"<br />
alt="Longest Labday" width="368"><p style="font-size: 12px;">Longest Labday:<br />18h </p></div></</li><br />
<li><div class="news"><img height="100px"<br />
src="https://static.igem.org/mediawiki/2010/b/b5/Freiburg10_Coffee_consumed_during_iGEM.png"<br />
alt="Coffee consumed during iGEM" width="184"><p style="font-size: 12px;">Coffee Consumed During iGEM:<br /> 2238 Cups</p></div></li><br />
<li><div class="news"><img heigth="100px"class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><p style="font-size: 12px;"><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h5>!Freiburg Bioware Statistics!</h5></a></p></div></li><br />
</ul><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<div height="290px"><br />
<!---Box on the upper right: BioBricks---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><img class="right" src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg_10_BioBrick_icon_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><h2>BioBricks</h2></a><br />
<p class="standard">Modularization and modification of the viral capsids for retargeting approaches and directed gene delivery of suicide genes resulted in many different BioBricks.<br> Click here to explore them.</p><br />
</div><br />
<br />
<!---Box on the lower left: Biosafety---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><img class="right" src="https://static.igem.org/mediawiki/2010/f/f9/Freiburg10_Danger_Virus_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><h2>Biosafety</h2></a><br />
<p class="standard">The aspect of biological safety was considered well and a risk assessment and security profile for the Adeno-associated Virus were created. Read more about Biosafety issues..</p><br />
<br />
</div><br />
<br />
<!---Box on the lower right: Notebook---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/95/Freiburg_10_Notebook_image_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<h2>Notebook</h2></a><br />
<p><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><span class="bold"><h5>Only cloning and cell culture? </h5></span></a><br /><br />
No! Besides <br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal"><span class="bold">BioBrick assembly</span></a>, flow cytometry analysis, quantification based methos via ELISA or Western Blot and cytotoxicity assays have been performed <a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Methods"><span class="bold"><h5>(go to Methods)</h5></span></p><br />
</a><br />
</div><br />
<br />
<br />
<!---Box on the lower right: Team---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_Virus_Logo_Small_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<h2>Team</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><h5>Fun and Science at the same time?</h5></a></a><br /><br />
Yes! The iGEM team Freiburg did not only share the lab bench, but as well some nice days <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Photo_Gallery"><span class="bold">canoeing</span></a> and initiated the photo contest <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Cuckoo_Clock"><span class="bold">Cuckoo Clock Competition</span></a></p><br />
</div><br />
</div><br />
<div class="clearfix"></div><br />
<br /><br />
<h1>Sponsors</h1><br />
<br />
<div class="map" height="1169px"><br />
<map name="Sponsoren_iGEM" id="Sponsoren_iGEM"><br />
<area shape="circle" coords="705,369,65" href="http://www.uni-potsdam.de/" target="_blank" alt="Uni Potsdam" title="Uni Potsdam" /><br />
<area shape="circle" coords="156,138,121" href="http://www.uni-freiburg.de/start-en.html?set_language=en" target="_blank" alt="Uni Freiburg" title="Uni Freiburg" /><br />
<area shape="rect" coords="304,8,815,269" href="http://www.bioss.uni-freiburg.de/cms/index.php#" target="_blank" alt="Bioss" title="Bioss" /><br />
<area shape="rect" coords="501,311,611,434" href="http://www.molbiotech.uni-freiburg.de/" target="_blank" alt="kuk.lab" title="kuk.lab" /><br />
<area shape="rect" coords="274,329,480,412" href="http://www.daad.de/en/index.html" alt="daad" target="_blank" title="daad" /><br />
<area shape="rect" coords="67,324,238,419" href="http://www.frias.uni-freiburg.de/" alt="frias" target="_blank" title="frias" /><br />
<area shape="rect" coords="95,534,376,686" href="http://www.roche.com/index.htm" target="_blank" alt="Roche" title="Roche" /><br />
<area shape="rect" coords="510,458,677,606" href="http://www.qiagen.com/default_qs.aspx" target="_blank" alt="Qiagen" title="Qiagen" /><br />
<area shape="rect" coords="477,671,708,752" href="http://www.starlab.de/int/?l=2" target="_blank" alt="Starlab" title="Starlab" /><br />
<area shape="rect" coords="551,799,787,861" href="http://www.peqlab.com" target="_blank" alt="peqlab" title="peqlab" /><br />
<area shape="rect" coords="291,801,494,859" href="http://www.eppendorf.com/int/?l=1&amp;action=start" target="_blank" alt="Eppendorf" title="Eppendorf" /><br />
<area shape="rect" coords="43,801,226,859" href="http://www.gilson.com/en/" target="_blank" alt="Gilson" title="Gilson" /><br />
<area shape="rect" coords="44,872,218,936" href="http://www.fermentas.de/index.php?&amp;language=en" target="_blank" alt="Fermentas" title="Fermentas" /><br />
<area shape="rect" coords="42,942,259,1002" href="http://www.home.agilent.com" target="_blank" alt="aigilent" title="aigilent" /><br />
<area shape="rect" coords="289,958,388,1041" href="http://www.avidity.com/" target="_blank" alt="Avidity" title="Avidity" /><br />
<area shape="rect" coords="412,960,566,1048" href="http://www.hiss-dx.de/hiss/index.php?id=43&amp;L=1" target="_blank" alt="Hiss" title="Hiss" /><br />
<area shape="rect" coords="591,981,795,1043" href="http://www.biozym.com/site/21/Produkte.aspx" target="_blank" alt="Biozym" title="Biozym" /><br />
<area shape="rect" coords="592,876,750,956" href="http://www.gatc-biotech.com" target="_blank" alt="GATC" title="GATC" /><br />
<area shape="rect" coords="296,886,540,948" href="http://www.geneart.com/" target="_blank" alt="Geneart" title="Geneart" /><br />
<area shape="rect" coords="495,1070,808,1151" href="http://www.atg-biosynthetics.com/" target="_blank" alt="ATG" title="ATG" /><br />
<area shape="rect" coords="242,1086,469,1154" href="http://www.purimex.com/" alt="purimex" target="_blank" title="purimex" /><br />
<area shape="rect" coords="45,1084,219,1154" href="http://www.dkfz.de/index.html" alt="dkfz" target="_blank" title="dkfz" /><br />
<area shape="rect" coords="46,1021,264,1075" href="http://www.mathworks.com" target="_blank" alt="Mathworks" title="Mathworks" /><br />
</map><br />
<img src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Sponsoren_Image_Map.gif" width="826" height="1169" border="0" alt="" title="" usemap="#Sponsoren_iGEM" /><br />
</div><br />
<br />
<br /><br />
<br /><br />
<center><a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
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<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-10-28T01:13:24Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<div class="virus"><img src="https://static.igem.org/mediawiki/2010/2/24/Freiburg10_rot250trans.gif" title="virus (3.5MB)" id="animated_virus"/></div><br />
<div class="div_home"><br />
<p><br />
Gene delivery using viral vectors holds great promise for the treatment of acquired and inherited diseases. The human Adeno-Associated Virus (AAV) is a small, non-pathogenic, single-stranded DNA virus gaining increasing attention being both versatile and effective. Taking current knowledge into account, we generated a recombinant, modularized, BioBrick-compatible AAV ‘Virus Construction Kit’. We provide parts for modified capsid proteins, targeting modules, tumor-specific promoters, and prodrug-activating enzymes as well as readily assembled vectors for gene delivery and production of non-replicative virus particles. The viral tropism is altered by N-terminal fusion or by loop replacement of the capsid proteins. Functionality of viruses constructed from our kit was demonstrated by fluorescent protein expression in infected cells and by prodrug-induced killing of tumor cells upon viral delivery of a thymidine kinase. Incorporating multiple layers of safety, we provide a general tool to the growing field of personalized medicine and demonstrate its use in tumor therapy.<br />
</p><br />
</div><br />
<br />
<!---Box on the upper left: Project Results---><br />
<div class="box_home"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project"><img class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><br />
</a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results"><h2>Project Results</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><h5>Highlights: </h5></a><br />
This Site is in Construction, later you will find more right here...<a href="https://2010.igem.org/Team:Freiburg_Bioware/Virus_Construction_Kit"><span class="bold">[more]</span></a></p><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Virus_Construction_Kit"><span class="bold"><h5>Heading:</h5></span></a><br />
This Site is in Construction, later on you will find more right here...<a href="https://2010.igem.org/Team:Freiburg_Bioware/Virus_Construction_Kit"><span class="bold">[Klick for more]</span></a></p><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold"><h5>Head-Line:</h5></span></a><br />
This Site is in Construction, later you will find more right here...<a href="https://2010.igem.org/Team:Freiburg_Bioware/Virus_Construction_Kit"><span class="bold">[Klick for more]</span></a></p><br />
</div><br />
<br />
<!---Big box on the left: Virus Construction Kit - Manual---><br />
<br />
<div class="box_long"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_Manual_Logo_small.png" id="ccc" /><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h2>Virus Construction Kit - The Manual</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h5>Heading:</h5></a><br />
Everything you need to know about AAV vectors!<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!--- News Ticker ---><br />
<br />
<div class="box_long box_long_news news_ticker"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h2>Statistics - In a Minute</h2></a><br />
<ul id="news" class="newsticker"><br />
<li><div class="news"><img class="right" src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_Number_of_Plasmids.png" id="ccc" /><p style="font-size: 12px;"><br />
What happened in the labduring iGEM? </p><br />
</div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_Longest_Wikipage.png"<br />
alt="Longest Wikipage" width="368"><p style="font-size: 12px;">Longest Wikipage:<br />with 222,795 bytes</p></div><br />
</li><br />
<li><div class="news"><img src="https://static.igem.org/mediawiki/2010/1/10/Freiburg10_Number_of_Biobricks.png"<br />
alt="Number of BioBricks" width="368"><p style="font-size: 12px;">Number of Biobricks:<br /> 116</p></div></li><br />
<li><div class="news"><img heigth="100px" src="https://static.igem.org/mediawiki/2010/3/3f/Freiburg10_Number_of_ordered_Oligo-Nucleotides.png"<br />
alt="Number of ordered Oligo-Nucleotides" width="184"><p style="font-size: 12px;">Number of Ordered Oligos:<br /> 193</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f3/Freiburg10_Minipreps_total.png"<br />
alt="Minipreps total" width="184"><p style="font-size: 12px;">Mini-Preps total:<br /> 1085</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f7/Freiburg10_Midipreps_total.png"<br />
alt="Midipreps total" width="368"><p style="font-size: 12px;">Midi-Preps total:<br /> 106</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d6/Freiburg10_Highest_Prep_concentration.png"<br />
alt="Highest Prep concentration (Midi)" width="368"><p style="font-size: 12px;">Highest Midi-Prep Concentration:<br />5045,6 ng/µL </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/af/Freiburg10_Number_of_Glyzerolstocks.png"<br />
alt="Number of Glycerolstocks" width="368"><p style="font-size: 12px;">Number of Glycerolstocks:<br /> 763 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/6/60/Freiburg10_Number_of_Team_members.png"<br />
alt="Number of Team members" width="368"><p style="font-size: 12px;">Number of Team members:<br /> 15 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/0/03/Freiburg10_Days_in_the_lab.png"<br />
alt="Days in the lab" width="368"><p style="font-size: 12px;">Days in the lab:<br /> 170 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_Longest_Labday.png"<br />
alt="Longest Labday" width="368"><p style="font-size: 12px;">Longest Labday:<br />18h </p></div></</li><br />
<li><div class="news"><img height="100px"<br />
src="https://static.igem.org/mediawiki/2010/b/b5/Freiburg10_Coffee_consumed_during_iGEM.png"<br />
alt="Coffee consumed during iGEM" width="184"><p style="font-size: 12px;">Coffee Consumed During iGEM:<br /> 2238 Cups</p></div></li><br />
<li><div class="news"><img heigth="100px"class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><p style="font-size: 12px;"><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h5>!Freiburg Bioware Statistics!</h5></a></p></div></li><br />
</ul><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<div height="290px"><br />
<!---Box on the upper right: BioBricks---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><img class="right" src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg_10_BioBrick_icon_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><h2>BioBricks</h2></a><br />
<p class="standard">Modularization and modification of the viral capsids for retargeting approaches and directed gene delivery of suicide genes resulted in many different BioBricks.<br> Click here to explore them.</p><br />
</div><br />
<br />
<!---Box on the lower left: Biosafety---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><img class="right" src="https://static.igem.org/mediawiki/2010/f/f9/Freiburg10_Danger_Virus_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><h2>Biosafety</h2></a><br />
<p class="standard">The aspect of biological safety was considered well and a risk assessment and security profile for the Adeno-associated Virus were created. Read more about Biosafety issues..</p><br />
<br />
</div><br />
<br />
<!---Box on the lower right: Notebook---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/95/Freiburg_10_Notebook_image_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<h2>Notebook</h2></a><br />
<p><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><span class="bold"><h5>Only cloning and cell culture? </h5></span></a><br /><br />
No! Besides <br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal"><span class="bold">BioBrick assembly</span></a>, flow cytometry analysis, quantification based methos via ELISA or Western Blot and cytotoxicity assays have been performed <a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Methods"><span class="bold"><h5>(go to Methods)</h5></span></p><br />
</a><br />
</div><br />
<br />
<br />
<!---Box on the lower right: Team---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_Virus_Logo_Small_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<h2>Team</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><h5>Fun and Science at the same time?</h5></a></a><br /><br />
Yes! The iGEM team Freiburg did not only share the lab bench, but as well some nice days <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Photo_Gallery"><span class="bold">canoeing</span></a> and initiated the photo contest <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Cuckoo_Clock"><span class="bold">Cuckoo Clock Competition</span></a></p><br />
</div><br />
</div><br />
<div class="clearfix"></div><br />
<br /><br />
<h1>Sponsors</h1><br />
<br />
<div class="map" height="1169px"><br />
<map name="Sponsoren_iGEM" id="Sponsoren_iGEM"><br />
<area shape="circle" coords="705,369,65" href="http://www.uni-potsdam.de/" target="_blank" alt="Uni Potsdam" title="Uni Potsdam" /><br />
<area shape="circle" coords="156,138,121" href="http://www.uni-freiburg.de/start-en.html?set_language=en" target="_blank" alt="Uni Freiburg" title="Uni Freiburg" /><br />
<area shape="rect" coords="304,8,815,269" href="http://www.bioss.uni-freiburg.de/cms/index.php#" target="_blank" alt="Bioss" title="Bioss" /><br />
<area shape="rect" coords="501,311,611,434" href="http://www.molbiotech.uni-freiburg.de/" target="_blank" alt="kuk.lab" title="kuk.lab" /><br />
<area shape="rect" coords="274,329,480,412" href="http://www.daad.de/en/index.html" alt="daad" target="_blank" title="daad" /><br />
<area shape="rect" coords="67,324,238,419" href="http://www.frias.uni-freiburg.de/" alt="frias" target="_blank" title="frias" /><br />
<area shape="rect" coords="95,534,376,686" href="http://www.roche.com/index.htm" target="_blank" alt="Roche" title="Roche" /><br />
<area shape="rect" coords="510,458,677,606" href="http://www.qiagen.com/default_qs.aspx" target="_blank" alt="Qiagen" title="Qiagen" /><br />
<area shape="rect" coords="477,671,708,752" href="http://www.starlab.de/int/?l=2" target="_blank" alt="Starlab" title="Starlab" /><br />
<area shape="rect" coords="551,799,787,861" href="http://www.peqlab.com" target="_blank" alt="peqlab" title="peqlab" /><br />
<area shape="rect" coords="291,801,494,859" href="http://www.eppendorf.com/int/?l=1&amp;action=start" target="_blank" alt="Eppendorf" title="Eppendorf" /><br />
<area shape="rect" coords="43,801,226,859" href="http://www.gilson.com/en/" target="_blank" alt="Gilson" title="Gilson" /><br />
<area shape="rect" coords="44,872,218,936" href="http://www.fermentas.de/index.php?&amp;language=en" target="_blank" alt="Fermentas" title="Fermentas" /><br />
<area shape="rect" coords="42,942,259,1002" href="http://www.home.agilent.com" target="_blank" alt="aigilent" title="aigilent" /><br />
<area shape="rect" coords="289,958,388,1041" href="http://www.avidity.com/" target="_blank" alt="Avidity" title="Avidity" /><br />
<area shape="rect" coords="412,960,566,1048" href="http://www.hiss-dx.de/hiss/index.php?id=43&amp;L=1" target="_blank" alt="Hiss" title="Hiss" /><br />
<area shape="rect" coords="591,981,795,1043" href="http://www.biozym.com/site/21/Produkte.aspx" target="_blank" alt="Biozym" title="Biozym" /><br />
<area shape="rect" coords="592,876,750,956" href="http://www.gatc-biotech.com" target="_blank" alt="GATC" title="GATC" /><br />
<area shape="rect" coords="296,886,540,948" href="http://www.geneart.com/" target="_blank" alt="Geneart" title="Geneart" /><br />
<area shape="rect" coords="495,1070,808,1151" href="http://www.atg-biosynthetics.com/" target="_blank" alt="ATG" title="ATG" /><br />
<area shape="rect" coords="242,1086,469,1154" href="http://www.purimex.com/" alt="purimex" target="_blank" title="purimex" /><br />
<area shape="rect" coords="45,1084,219,1154" href="http://www.dkfz.de/index.html" alt="dkfz" target="_blank" title="dkfz" /><br />
<area shape="rect" coords="46,1021,264,1075" href="http://www.mathworks.com" target="_blank" alt="Mathworks" title="Mathworks" /><br />
</map><br />
<img src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Sponsoren_Image_Map.gif" width="826" height="1169" border="0" alt="" title="" usemap="#Sponsoren_iGEM" /><br />
</div><br />
<br />
<br /><br />
<br /><br />
<center><a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
</a></center><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/NoteBook/LabjournalTeam:Freiburg Bioware/NoteBook/Labjournal2010-10-28T01:05:35Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_notebook}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
<br />
<html><br />
<div style="height: 850px; width: 964px; margin: 15px 15px 15px 15px;"<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March"><br />
<h2 style="margin-left: 5px;">March</h2><br />
<p class="boxes_labjournal_day">Labday 1</p><br />
<img src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg_10_labjournal_march_v1.JPG" class="image_labjournal" title="Our project starts with first sequence analysis."/><br />
<p class="boxes_labjournal_text">...watching gene sequences...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April"><br />
<h2 style="margin-left: 5px;">April</h2><br />
<p class="boxes_labjournal_day">Labday 2- 5</p><br />
<img src="https://static.igem.org/mediawiki/2010/f/fc/Freiburg_10_labjournal_april_v1.JPG" class="image_labjournal" title="Scanning for iGEM restriction sites: <br />
Vector plasmid; Thymidine kinase ;Cytosindeaminase" /><br />
<p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May"><br />
<h2 style="margin-left: 5px;">May</h2><br />
<p class="boxes_labjournal_day">Labday 6- 17</p><br />
<img src="https://static.igem.org/mediawiki/2010/0/0f/Freiburg_10_labjournal_may_v1.JPG" class="image_labjournal" title="We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt." /><br />
<p class="boxes_labjournal_text">...theoretical cloning...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June"><br />
<h2 style="margin-left: 5px;">June</h2><br />
<p class="boxes_labjournal_day">Labday 18- 45</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/37/Freiburg_10_labjournal_june_v1.jpg" class="image_labjournal" title="<br />
We conducted our first site-directed mutagenesis. First cell culture steps: Splitting and seeding of AAV 293 and HT1080 cells; Calcium phosphate transfection; Transduction with virus particles containing YFP encoding sequence; Microscopy: Fluorescent transduced HT1080 cells. We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared. Theoretical studies of the AAV structure: Impressions" /><br />
<p class="boxes_labjournal_text">...first transduced cells...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July"><br />
<h2 style="margin-left: 5px;">July</h2><br />
<p class="boxes_labjournal_day">Labday 46- 75</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8d/Freiburg_10_labjournal_july_v1.JPG" class="image_labjournal" title="We developed a plan for modifying the surface exposed loops of the virus capsid.<br />
For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI. BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix. Further on we received first quantitative transduction results via flow cytometry. mGMK_TK30 was successfully cloned into the vector plasmid." /><br />
<p class="boxes_labjournal_text">...organizing the lab...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August"><br />
<h2 style="margin-left: 5px;">August I</h2><br />
<p class="boxes_labjournal_day">Labday 76- 92</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/Freiburg_10_labjournal_august1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...cloning parts...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2"><br />
<h2 style="margin-left: 5px;">August II</h2><br />
<p class="boxes_labjournal_day">Labday 93- 106</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg_10_labjournal_august2_v1.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...assembled vector plasmid is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September"><br />
<h2 style="margin-left: 5px;">September I</h2><br />
<p class="boxes_labjournal_day">Labday 107- 123</p><br />
<img src="https://static.igem.org/mediawiki/2010/2/25/Freiburg_10_labjournal_september1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...simply producing BioBricks...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2"><br />
<h2 style="margin-left: 5px;">September II</h2><br />
<p class="boxes_labjournal_day">Labday 124- 135</p><br />
<img src="https://static.igem.org/mediawiki/2010/1/18/Freiburg_10_labjournal_october2_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...21x mutated RepCap is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October"><br />
<h2 style="margin-left: 5px;">October I</h2><br />
<p class="boxes_labjournal_day">Labday 136- 149</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/e0/Freiburg_10_labjournal_october_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...testing BioBricks by virus production...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2"><br />
<h2 style="margin-left: 5px;">October II</h2><br />
<p class="boxes_labjournal_day">Labday 150- 166</p><br />
<img src="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_Oktober.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Capsid modification works...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November"><br />
<h2 style="margin-left: 5px;">November</h2><br />
<p class="boxes_labjournal_day">Labday 166- 170</p><br />
<img src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_November.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Jamboree!!!...</p><br />
</a><br />
</div><br />
<br />
</div><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/NoteBook/LabjournalTeam:Freiburg Bioware/NoteBook/Labjournal2010-10-28T01:04:29Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_notebook}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
<br />
<html><br />
<div style="height: 850px; width: 964px; margin: 15px 15px 15px 15px;"<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March"><br />
<h2 style="margin-left: 5px;">March</h2><br />
<p class="boxes_labjournal_day">Labday 1</p><br />
<img src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg_10_labjournal_march_v1.JPG" class="image_labjournal" title="Our project starts with first sequence analysis."/><br />
<p class="boxes_labjournal_text">...watching gene sequences...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April"><br />
<h2 style="margin-left: 5px;">April</h2><br />
<p class="boxes_labjournal_day">Labday 2- 5</p><br />
<img src="https://static.igem.org/mediawiki/2010/f/fc/Freiburg_10_labjournal_april_v1.JPG" class="image_labjournal" title="Scanning for iGEM restriction sites: <br />
Vector plasmid; Thymidine kinase ;Cytosindeaminase" /><br />
<p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May"><br />
<h2 style="margin-left: 5px;">May</h2><br />
<p class="boxes_labjournal_day">Labday 6- 17</p><br />
<img src="https://static.igem.org/mediawiki/2010/0/0f/Freiburg_10_labjournal_may_v1.JPG" class="image_labjournal" title="We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt." /><br />
<p class="boxes_labjournal_text">...theoretical cloning...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June"><br />
<h2 style="margin-left: 5px;">June</h2><br />
<p class="boxes_labjournal_day">Labday 18- 45</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/37/Freiburg_10_labjournal_june_v1.jpg" class="image_labjournal" title="<br />
We conducted our first site-directed mutagenesis. First cell culture steps: Splitting and seeding of AAV 293 and HT1080 cells; Calcium phosphate transfection; Transduction with virus particles containing YFP encoding sequence; Microscopy: Fluorescent transduced HT1080 cells. We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared. Theoretical studies of the AAV structure: Impressions" /><br />
<p class="boxes_labjournal_text">...first transduced cells...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July"><br />
<h2 style="margin-left: 5px;">July</h2><br />
<p class="boxes_labjournal_day">Labday 46- 75</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8d/Freiburg_10_labjournal_july_v1.JPG" class="image_labjournal" title="We developed a plan for modifying the surface exposed loops of the virus capsid.<br />
For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI. BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix. Further on we received first quantitative transduction results via flow cytometry. mGMK_TK30 was successfully cloned into the vector plasmid." /><br />
<p class="boxes_labjournal_text">...organizing the lab...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August"><br />
<h2 style="margin-left: 5px;">August I</h2><br />
<p class="boxes_labjournal_day">Labday 76- 92</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/Freiburg_10_labjournal_august1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...cloning parts...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2"><br />
<h2 style="margin-left: 5px;">August II</h2><br />
<p class="boxes_labjournal_day">Labday 93- 106</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg_10_labjournal_august2_v1.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...assembled vector plasmid is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September"><br />
<h2 style="margin-left: 5px;">September I</h2><br />
<p class="boxes_labjournal_day">Labday 107- 123</p><br />
<img src="https://static.igem.org/mediawiki/2010/2/25/Freiburg_10_labjournal_september1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...simply producing BioBricks...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2"><br />
<h2 style="margin-left: 5px;">September II</h2><br />
<p class="boxes_labjournal_day">Labday 124- 135</p><br />
<img src="https://static.igem.org/mediawiki/2010/1/18/Freiburg_10_labjournal_october2_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...21x mutated RepCap is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October"><br />
<h2 style="margin-left: 5px;">October I</h2><br />
<p class="boxes_labjournal_day">Labday 136- 149</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/e0/Freiburg_10_labjournal_october_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...testing BioBricks by virus production...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2"><br />
<h2 style="margin-left: 5px;">October II</h2><br />
<p class="boxes_labjournal_day">Labday 150- 166</p><br />
<img src="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_Oktober.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Capsid modification works...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November"><br />
<h2 style="margin-left: 5px;">November</h2><br />
<p class="boxes_labjournal_day">Labday 166- 170</p><br />
<img src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_November.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Jemboree!!!...</p><br />
</a><br />
</div><br />
<br />
</div><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/File:Freiburg10_November.pngFile:Freiburg10 November.png2010-10-28T01:03:17Z<p>Achim: </p>
<hr />
<div></div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/NoteBook/LabjournalTeam:Freiburg Bioware/NoteBook/Labjournal2010-10-28T00:58:14Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_notebook}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
<br />
<html><br />
<div style="height: 850px; width: 964px; margin: 15px 15px 15px 15px;"<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March"><br />
<h2 style="margin-left: 5px;">March</h2><br />
<p class="boxes_labjournal_day">Labday 1</p><br />
<img src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg_10_labjournal_march_v1.JPG" class="image_labjournal" title="Our project starts with first sequence analysis."/><br />
<p class="boxes_labjournal_text">...watching gene sequences...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April"><br />
<h2 style="margin-left: 5px;">April</h2><br />
<p class="boxes_labjournal_day">Labday 2- 5</p><br />
<img src="https://static.igem.org/mediawiki/2010/f/fc/Freiburg_10_labjournal_april_v1.JPG" class="image_labjournal" title="Scanning for iGEM restriction sites: <br />
Vector plasmid; Thymidine kinase ;Cytosindeaminase" /><br />
<p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May"><br />
<h2 style="margin-left: 5px;">May</h2><br />
<p class="boxes_labjournal_day">Labday 6- 17</p><br />
<img src="https://static.igem.org/mediawiki/2010/0/0f/Freiburg_10_labjournal_may_v1.JPG" class="image_labjournal" title="We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt." /><br />
<p class="boxes_labjournal_text">...theoretical cloning...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June"><br />
<h2 style="margin-left: 5px;">June</h2><br />
<p class="boxes_labjournal_day">Labday 18- 45</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/37/Freiburg_10_labjournal_june_v1.jpg" class="image_labjournal" title="<br />
We conducted our first site-directed mutagenesis. First cell culture steps: Splitting and seeding of AAV 293 and HT1080 cells; Calcium phosphate transfection; Transduction with virus particles containing YFP encoding sequence; Microscopy: Fluorescent transduced HT1080 cells. We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared. Theoretical studies of the AAV structure: Impressions" /><br />
<p class="boxes_labjournal_text">...first transduced cells...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July"><br />
<h2 style="margin-left: 5px;">July</h2><br />
<p class="boxes_labjournal_day">Labday 46- 75</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8d/Freiburg_10_labjournal_july_v1.JPG" class="image_labjournal" title="We developed a plan for modifying the surface exposed loops of the virus capsid.<br />
For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI. BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix. Further on we received first quantitative transduction results via flow cytometry. mGMK_TK30 was successfully cloned into the vector plasmid." /><br />
<p class="boxes_labjournal_text">...organizing the lab...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August"><br />
<h2 style="margin-left: 5px;">August I</h2><br />
<p class="boxes_labjournal_day">Labday 76- 92</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/Freiburg_10_labjournal_august1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...cloning parts...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2"><br />
<h2 style="margin-left: 5px;">August II</h2><br />
<p class="boxes_labjournal_day">Labday 93- 106</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg_10_labjournal_august2_v1.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...assembled vector plasmid is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September"><br />
<h2 style="margin-left: 5px;">September I</h2><br />
<p class="boxes_labjournal_day">Labday 107- 123</p><br />
<img src="https://static.igem.org/mediawiki/2010/2/25/Freiburg_10_labjournal_september1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...simply producing BioBricks...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2"><br />
<h2 style="margin-left: 5px;">September II</h2><br />
<p class="boxes_labjournal_day">Labday 124- 135</p><br />
<img src="https://static.igem.org/mediawiki/2010/1/18/Freiburg_10_labjournal_october2_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...21x mutated RepCap is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October"><br />
<h2 style="margin-left: 5px;">October I</h2><br />
<p class="boxes_labjournal_day">Labday 136- 149</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/e0/Freiburg_10_labjournal_october_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...testing BioBricks by virus production...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2"><br />
<h2 style="margin-left: 5px;">October II</h2><br />
<p class="boxes_labjournal_day">Labday 150- 166</p><br />
<img src="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_Oktober.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Capsid modification works...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November"><br />
<h2 style="margin-left: 5px;">November</h2><br />
<p class="boxes_labjournal_day">Labday 166- 170</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Jemboree!!!...</p><br />
</a><br />
</div><br />
<br />
</div><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/File:Freiburg10_Oktober.pngFile:Freiburg10 Oktober.png2010-10-28T00:56:46Z<p>Achim: </p>
<hr />
<div></div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/FilelistTeam:Freiburg Bioware/Filelist2010-10-28T00:35:31Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>AAAAAH</h2><br />
[[Image:Freiburg10_Organisation_cap_proteins.png]]<br />
[[Image:Freiburg10 Organisation rep proteins.png]]<br />
<br />
<h2>eGFP</h2><br />
[[Media:Freiburg10_eGFP.png]]<br />
[[Media:Freiburg10_CD.png]]<br />
[[Media:Freiburg10_pathway_CD.png]]<br />
[[Media:Freiburg10_CFP_spectrum]]<br />
[[Media:Freiburg10_CFP_fluorophor]]<br />
[[Media:Freiburg10_CFP_architecture]]<br />
<br />
<br />
<br />
<h2>Time lapse</h2><br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
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[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Structure Modeling</h2><br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
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[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
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[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
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[[Media:Freiburg10_AAV_slice.gif]]<br />
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[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
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[[Media:Freiburg10_PleaseWait.gif]]<br />
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[[Media:Freiburg10_AAV_assembly_600px.png]]<br />
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[[Media:Freiburg10_AAV_assembly_400px.png]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_scheme_400px.png]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]] **neues Bild hochladen!**<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
[[Media:Freiburg10_CD.jpg]]<br />
<br />
[[Media:Freiburg10_TK.jpg]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
[[Media:Freiburg10_ELISA.jpg]]<br />
<br />
[[Media:Freiburg10_ELISA_plate.jpg]]<br />
<br />
[[Media:Freiburg10_akta.jpg]]<br />
<br />
[[Media:Freiburg10_his_column.jpg]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 mVenusDrop.PNG]]<br />
<br />
[[Media:Freiburg10 qPCR DARPin.PNG]]<br />
<br />
[[Media:Freiburg10 FACS1.PNG]]<br />
<br />
[[Media:Freiburg10 FACS2.PNG]]<br />
<br />
[[Media:Freiburg10 FACS3.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP1Insnew.PNG]]<br />
<br />
[[Media:Freiburg10 qPCR VP2Fus.PNG]]<br />
<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]<br />
<br />
<h2>ITR Diary </h2><br />
<br />
<br />
<h2> Targeting: Flow Cytometry: DARPin, Affi (HSPGKO) </h2><br />
<br />
<br />
[[Media:Freiburg10 FACS DARPin Affi HSPGKO A431.png]]<br />
<br />
[[Media:Freiburg10_FACS DARPin Affi HSPGKO HT1080.png]]<br />
<br />
[[Media:Freiburg10_FlowCytometry Diagramm DARPin Affi.png]]</div>Achimhttp://2010.igem.org/File:Freiburg10_Organisation_rep_proteins.pngFile:Freiburg10 Organisation rep proteins.png2010-10-28T00:34:41Z<p>Achim: </p>
<hr />
<div></div>Achimhttp://2010.igem.org/File:Freiburg10_Organisation_cap_proteins.pngFile:Freiburg10 Organisation cap proteins.png2010-10-28T00:33:04Z<p>Achim: </p>
<hr />
<div></div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/Project_DescriptionTeam:Freiburg Bioware/Project/Project Description2010-10-28T00:10:55Z<p>Achim: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<br />
<html><br />
<div class=WordSection1><br />
<br />
<div style='border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 0cm 1.0pt 0cm;<br />
margin-left:21.6pt;margin-right:0cm'><br />
<br />
<p class=MsoTocHeading style='margin-left:0cm;text-indent:0cm'><a<br />
name="_Toc274911361"></a><a name="_Toc275994040"><span lang=DE>Contents</span></a></p><br />
<br />
</div><br />
<br />
<p class=MsoToc1><a href="#_Toc275994657"><span lang=DE>Overview</span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>.. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>1</span></a></p><br />
<br />
<p class=MsoToc2><a href="#_Toc275994658"><span lang=DE>The Experimental System</span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>.. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>1</span></a></p><br />
<br />
<p class=MsoToc2><a href="#_Toc275994659"><span lang=DE>Layers of specificity</span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>2</span></a></p><br />
<br />
<p class=MsoToc1><a href="#_Toc275994660">Introduction to Adeno-Associated<br />
Virus Serotype 2<span lang=DE style='color:windowtext;display:none;text-decoration:<br />
none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>4</span></a></p><br />
<br />
<p class=MsoToc2><a href="#_Toc275994661">Biology of the AAV-2<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>4</span></a></p><br />
<br />
<p class=MsoToc3><a href="#_Toc275994662">Genomic organization<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>4</span></a></p><br />
<br />
<p class=MsoToc3><a href="#_Toc275994663">Replication<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>5</span></a></p><br />
<br />
<p class=MsoToc3><a href="#_Toc275994664">Integration<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>11</span></a></p><br />
<br />
<p class=MsoToc3><a href="#_Toc275994665">Rescue<span lang=DE style='color:<br />
windowtext;display:none;text-decoration:none'> </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>12</span></a></p><br />
<br />
<p class=MsoToc3><a href="#_Toc275994666">Rep proteins<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>12</span></a></p><br />
<br />
<p class=MsoToc3><a href="#_Toc275994667">VP proteins<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>16</span></a></p><br />
<br />
<p class=MsoToc3><a href="#_Toc275994668">Trafficking<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'> </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>23</span></a></p><br />
<br />
<p class=MsoToc2><a href="#_Toc275994669">Helper Genes<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>27</span></a></p><br />
<br />
<p class=MsoToc2><a href="#_Toc275994670">Recombinant Viruses and Mosaic<br />
Viruses<span lang=DE style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>29</span></a></p><br />
<br />
<p class=MsoToc2><a href="#_Toc275994671"><span lang=DE>Gene Therapy</span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>30</span></a></p><br />
<br />
<p class=MsoToc2><a href="#_Toc275994672">Immune Response<span lang=DE<br />
style='color:windowtext;display:none;text-decoration:none'>. </span><span<br />
lang=DE style='color:windowtext;display:none;text-decoration:none'>31</span></a></p><br />
<br />
<p class=MsoNormal><span lang=DE>&nbsp;</span></p><br />
<br />
<div style='border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 0cm 1.0pt 0cm'><br />
<br />
<h1 style='margin-left:0cm;text-indent:0cm'><span lang=DE>&nbsp;</span></h1><br />
<br />
<h1 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994657"><span<br />
lang=DE>Overview</span></a></h1><br />
<br />
</div><br />
<br />
<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994658"></a><a<br />
name="_Toc275994041"><span lang=DE>The Experimental System</span></a></h2><br />
<br />
<p class=MsoNormal>Therapy using viral vectors is an promising approac. In an<br />
first step the plasmids of the AAV-2 Helper-free System were genetically<br />
modifyed by converting it into BioBricks and inserting of targeting molecules<br />
into the constructs. These plasmids were then used to transfect the producer<br />
cell line AAV-293. After an incubation of three days the viral vectors were<br />
harvested and used to transduce different target cells. The succesful<br />
transduction can then for example be measured by detecting the fluorescence of<br />
fluorescent proteins in the target cells</p><br />
<br />
<p class=MsoNormal>The majority of the modifications that were introduced into<br />
the viral vector aimed to allow differential targeting of tumor cell over<br />
healthy off-target cells.</p><br />
<br />
<p class=MsoNormal><img width=471 height=575 id="Picture 56"<br />
src="Introductionwiki-Dateien/image001.png"<br />
alt="Beschreibung: X:\users\FreiGem\iGEM2010\Volker\g16268.png"></p><br />
<br />
<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994659"></a><a<br />
name="_Toc275994042"><span lang=DE>Layers of specificity</span></a></h2><br />
<br />
<p class=MsoNormal>Employment of viral vectors for means of therapy is idea in<br />
the context of personalized medicie that gets more and more interest. In such<br />
applications the reduction of side effects and the safety of the patient in<br />
general is of the highest priority.</p><br />
<br />
<p class=MsoNormal>In order to satisfy this requirement we designed our Therapy<br />
Vector with several layers of Specificity:</p><br />
<br />
<p class=MsoNormal>The targeting of the viral vector towards the desired target<br />
cell (e.g. tumor cells) is the basic idea behind the emplyment of viral vectors<br />
for therapeutical means. There for the natural tropismn has to be knocked down<br />
and a desired tropism has to be introduced that allows differential targeting<br />
of pathological but not of off-target cells. To fulfill this mission our Virus<br />
Construction Kit offers you different solutions.</p><br />
<br />
<p class=MsoNormal>Off-target cells that were transduced by mistake can be<br />
preserved from an undesired therapy effect when the therapeutic gene is<br />
controley by a tissue specific promoter. For this mean a promoter has to be<br />
used that is as specific for the pathological tissue as possible. We included<br />
the human telomerase promoter (<a<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K404106">phTERT</a>)<br />
which is often activated in tumor cells and is there for able to allow<br />
differential experssion of a therapeutic geneproduct in pathological cells.</p><br />
<br />
<p class=MsoNormal>For reasons of safety Therapeutic vector do not directly<br />
trigger appoptosis in the successfully targeted cells. To include one further<br />
layer of specificity and safety we decided to arm our therapy vector with<br />
different prodrug convertases. Neither the single application of the harmless<br />
prodrug nor the single expression of the convertase has a noteworthy effect of<br />
the transduced cell. Only in cells that express the prodrug convertase and have<br />
a sufficient cytoplasmatic concentration of the belonging prodrug apoptosis is<br />
triggered. This dependency of the therapy on a prodrug can be employed to<br />
protect tissues or other persons that could come in contact with the<br />
therapeutical vector. This aspect was specially inportant for the development<br />
of a viral vector that is able to infect humans in the context of a<br />
undergraduate project for the iGEM competition. Therefor this approach gained<br />
our preference over other possibly equivalent arming possibilities described in<br />
the tumor therapy with viral vectors.</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal><img border=0 width=463 height=512 id="Picture 57"<br />
src="Introductionwiki-Dateien/image002.png"<br />
alt="Beschreibung: X:\users\FreiGem\iGEM2010\Volker\g16404.png"></p><br />
<br />
</div><br />
<br />
<span lang=DE style='font-size:11.0pt;line-height:150%;font-family:"Calibri","sans-serif"'><br<br />
clear=all style='page-break-before:always'><br />
</span><br />
<br />
<div class=WordSection2><br />
<br />
<div style='border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 0cm 1.0pt 0cm'><br />
<br />
<h1 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994660"></a><a<br />
name="_Toc275994043">Introduction to </a>Adeno-Associated Virus Serotype 2</h1><br />
<br />
</div><br />
<br />
<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994661"></a><a<br />
name="_Toc275994044"></a><a name="_Toc274911362">Biology of the AAV-2</a><a<br />
name="_Toc274911363"></a></h2><br />
<br />
<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994662"></a><a<br />
name="_Toc275994045">Genomic organization</a></h3><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:4.8pt;margin-right:<br />
4.8pt'><br />
<tr style='height:191.45pt'><br />
<td width=568 valign=top style='width:426.05pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt;height:191.45pt'><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a<br />
name="_Toc274911364"><img border=0 width=524 height=269 id="Grafik 154"<br />
src="Introductionwiki-Dateien/image003.png"></a></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a<br />
name="_Ref275946742">Figure </a>1: Genomic organization of the wt-AAV-2. The<br />
inverted terminal repeats (ITRs) flank the two open reading frames (ORFs).<br />
The four-nonstructural proteins encoded from the <i>rep</i> gene are driven<br />
by the p5 and p19 promoters, whereas the structural Cap proteins are<br />
regulated by the p40 promoter. Additionally, the Assembly Activating Protein<br />
(AAP) was found recently within the <i>cap</i> gene.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>The Adeno-associated virus serotype-2 (AAV-2) genome is a linear,<br />
single-stranded (ss) 4675 bp DNA virus. Due to its small size, gene genomic<br />
organization is condensed and gene regulation is complex. The viral nucleotide<br />
sequence consist of two open reading frames (ORFs) coding for Rep- and Cap<br />
proteins and are flanked on either side by identical inverted terminal repeat<br />
(ITR) structures which are palindromic and form hairpin structures (Srivastava et al. 1983). </p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>The ITRs serve as primers for the host cells’ DNA polymerase,<br />
which converts the single-stranded virus genome into double-stranded DNA (ds<br />
DNA) as a part of the viruses’ replicative cycle. They also play important<br />
roles in viral genome integration into and rescue from the hosts genome, the<br />
formation of concatamers in the host cell nucleus and encapsidation of the<br />
viral genome into preforemd capsids (Berns 1990). Due to these essential functions, the ITR structures cannot be deleted from<br />
a viral vector and need to be delivered in <i>cis.</i></p><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994046">Organization<br />
of the Inverted Terminal </a>Repeat Structure</h4><br />
<br />
<p class=MsoNormal><span style='color:black'>The inverted terminal repeat<br />
structures can be subdivided into several palindromic motives: A and A’ form a<br />
stem loop which encases B and B’ as well as C and C’. Those motives form both<br />
arms of the T-shaped structure. The functional motives on the ITR are two<br />
regions that bind Rep 68/78, called Rep-binding elements (RBE on the stem and<br />
RBE’ on the B arm) and the terminal resolution site (trs) in which the rep<br />
proteins introduce single-stranded nicks. The 3’ OH end of the A motive acts as<br />
a primer for DNA replication </span><span<br />
style='color:black'>(Im &amp; Muzyczka 1990)</span><span style='color:black'> </span><span style='color:black'>(Lusby et al. 1980)</span><span style='color:black'>.</span></p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:293.05pt'><br />
<td width=520 valign=top style='width:390.3pt;padding:0cm 5.4pt 0cm 5.4pt;<br />
height:293.05pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><img border=0 width=512 height=384 id="Grafik 11"<br />
src="Introductionwiki-Dateien/image004.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 2: Organization of the ITRs, which are the only <i>cis</i>-required element in viral<br />
genome integration and replication. </p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994663"></a><a<br />
name="_Toc275994047">Replication</a></h3><br />
<br />
<p class=MsoNormal>The 3’ OH end of the viral DNA folds onto itself as part of<br />
the inverted terminal repeat (ITR) structure and thus serves as a primer for<br />
elongation by the host cell’s DNA polymerase. The polymerases strand<br />
displacement activity unfolds the opposite ITR structure and elongation<br />
continues until the 5’ template end is reached <span style='color:black'> </span><span style='color:black'>(Lusby et al. 1980)</span>.</p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr><br />
<td width=614 valign=top style='width:460.5pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><img border=0 width=588 height=413 id="Grafik 369"<br />
src="Introductionwiki-Dateien/image005.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 3: Figure 3: Schematic overview of AAV-2 replication.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>The remaining hairpin structure that served as the origin of<br />
replication then acts as a target for the Rep 68/78 protein: It binds to the<br />
Rep-binding site (RBS) and unwinds the double-stranded DNA in a way that the<br />
terminal resolution site (trs) is being displayed in a single-stranded form on<br />
a stem loop. This enables the endonuclease catalytic domain of the Rep protein<br />
to introduce a nick of the parental strand at this site, which in turn serves<br />
as a new primer for DNA polymerase. The polymerase resolves the hairpin structure<br />
through strand displacement and copies the remaining end of the parental strand<span<br />
style='color:black'> </span><span<br />
style='color:black'>(Im &amp; Muzyczka 1990)</span> .</p><br />
<br />
<p class=MsoNormal>Sometimes, nicking does not occur after polymerases have<br />
partially copied the virus DNA. In this case, the newly synthesized 3’ end acts<br />
as a primer and the host cell’s DNA polymerase copies the whole sequence once<br />
again, displacing the ITR strands in the middle of the sequence. This leads to<br />
a dsDNA containing the whole virus genome twice, called a duplex dimer (DD).<br />
Those dimers can be resolved back to duplex monomers (DM) by the Rep proteins</p><br />
<br />
<p class=MsoNormal>After replication, the dsDNA separates again forming new<br />
ssDNA in (+) and (-) polarity with hairpin structures at its ends. The Rep<br />
40/52 proteins are involved in this process. Newly synthesized copies are<br />
either encapsidated into virus capsids or replicated again (Gonçalves, 2005a). Double-stranded genomes are formed as well through annealing of (+) - and<br />
(-) single strands. Both mechanisms occur during infection and contribute to<br />
transgene expression (Schultz &amp; Chamberlain 2008). </p><br />
<br />
<p class=MsoNormal>If the double stranded virus DNA exists in an episomal form<br />
inside the nucleus, it tends to form linear as well as circular concatamers,<br />
which are formed by ligation of duplex monomers(Schultz &amp; Chamberlain 2008). </p><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994048">Viral<br />
promoters</a></h4><br />
<br />
<p class=MsoNormal>Three viral promoters are coordinating gene expression in<br />
the wildtype AAV-2. Each promoter regulates different open reading frames<br />
(ORFs) of regulatory proteins (p5 and p19 promoter) and structural proteins<br />
(p40 promoter). A general overview is provided in<b>Error! Reference source not<br />
found.</b>. p5 and p19 promoters are repressed in absence of helper proteins<br />
provided by Ad or HSV whereas transactivation of p5 and p19 occurs in presence<br />
of helper viruses. Furthermore, the larger Rep proteins activate the p40<br />
promoter. Since overexpression of Rep78 leads to cell cycle arrest, high levels<br />
of Rep78/68 lead to repression of the p5 promoter. </p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:373.8pt'><br />
<td width=379 valign=top style='width:284.5pt;padding:0cm 5.4pt 0cm 5.4pt;<br />
height:373.8pt'><br />
<p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;<br />
text-indent:0cm;page-break-after:avoid'><img border=0 width=363 height=432<br />
id="Grafik 1226" src="Introductionwiki-Dateien/image006.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a<br />
name="_Ref275633280"></a><a name="_Ref275633331">Figure </a>4: Regulation of the viral promoters located within the t AAV-2 genome. In the absence of<br />
helper viruses, gene expression is suppressed, whereas activation of p5 and<br />
p19 occurs in the presence of helper proteins by interacting of Rep proteins<br />
with cellular and helper proteins.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal align=center style='text-align:center'>&nbsp;</p><br />
<br />
<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994049"></a><a<br />
name="_Ref275645430">p5 promoter</a></h5><br />
<br />
<p class=MsoNormal>The p5 promoter, located downstream of the <i>rep</i> and <i>cap</i><br />
ORF (Figure 5: The p5 promoter of the wtAAV-2 is located upstream of the rep<br />
and cap ORF and contains several elements, which interact with Rep and<br />
endogenous proteins.)of the wtAAV-2, regulates gene expression of the two larger<br />
non-structural proteins Rep 78 and Rep 68 that are essential in genome<br />
replication and viral genome integration into several hotspots of the human<br />
chromosome. </p><br />
<br />
<p class=MsoNormal>Several binding elements for cellular and viral proteins<br />
involved in regulation can be found in the p5 promoter (Figure 5) therefore<br />
playing an important role in gene transcription, integration and replication,<br />
dependent on the presence or absence of helper viruses such as adenovirus (Ad)<br />
or herpes simplex virus (HSV) (Murphy et al. 2007). Besides regulation of gene expression, the p5 integration efficient<br />
element (p5IEE) containing the rep binding element (RBE) and a terminal<br />
resolution site (trs) is responsible for mediating site specific integration<br />
into the human genome (Philpott et al. 2002). </p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:203.8pt'><br />
<td width=578 valign=top style='width:433.85pt;padding:0cm 5.4pt 0cm 5.4pt;<br />
height:203.8pt'><br />
<p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;<br />
text-indent:0cm;page-break-after:avoid'><img border=0 width=566 height=235<br />
id="Grafik 1286" src="Introductionwiki-Dateien/image007.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a<br />
name="_Ref275633988"></a><a name="_Ref275633992">Figure </a>5: The p5 promoter of the wtAAV-2 is located upstream of the rep and cap ORF and contains<br />
several elements, which interact with Rep and endogenous proteins.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal><span lang=EN-GB>&nbsp;</span></p><br />
<br />
<p class=MsoNormal>Containing two consensus sequences for binding immediate<br />
early E1A gene product from adenoviruses (Chang et al. 1989), p5 promoter is<br />
transactivated in the presence of helper viruses whereas suppression occurs in<br />
absence of adenoviral proteins by low levels of Rep proteins (Beaton et al. 1989). Regulating of Rep78/68 by its negative feedback loop is critical<br />
since overexpression leads to cell cycle arrest in the S-phase (Berthet et al. 2005) and suppression of cellular promoters (Jing et al. 2001).</p><br />
<br />
<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994050">p5<br />
TATA-less promoter</a></h5><br />
<br />
<p class=MsoNormal>In contrast to the natural location of the p5 promoter, the<br />
iGEM team Freiburg 2010 provides the RepCap plasmid with a relocated p5 promoter<br />
downstream of the <i>RepCap</i> genes (Figure 6). Additionally the p5 promoter<br />
lacks the TATA box element (AVIGEN 1997)<b>. </b>Those modifications result in an attenuated expression of the larger<br />
Rep proteins therefore leading to normal transcription of the Rep proteins<br />
driven by p19 promoter and enhanced expression of the Cap proteins, which are<br />
under the control of the p40 promoter. Additionally, removing the p5 promoter<br />
downstream of the <i>RepCap</i> genes and deletion of the TATA box eliminates<br />
contamination with wtAAVs. Hence, alteration of the p5 promoter is useful for<br />
enhanced production of recombinant viral particles attenuating repression of<br />
Rep78/68 and improving gene transcription of the capsid proteins and Rep<br />
proteins involved in genome packaging. </p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:214.9pt'><br />
<td width=316 valign=top style='width:236.7pt;padding:0cm 5.4pt 0cm 5.4pt;<br />
height:214.9pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><img border=0 width=300 height=243 id="Grafik 1289"<br />
src="Introductionwiki-Dateien/image008.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a<br />
name="_Ref275634090">Figure </a>6: p5 TATA-less promoter is located<br />
downstream of the rep and cap ORF.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994051"></a><a<br />
name="_Ref275645443"></a><a name="_Ref275628172">p19</a> promoter</h5><br />
<br />
<p class=MsoNormal>p19 promoter drives gene expression of the smaller Rep proteins<br />
Rep52 and Rep40. In absence of a helper virus infection the promoter is<br />
inactive by repression of all four Rep proteins, but is transactivated by<br />
interaction of both the Sp1 site and Rep protein Rep78/68 bound to the Rep<br />
binding element (RBE) (Lackner &amp; Muzyczka 2002). By forming a DNA loop (Pereira &amp; Muzyczka 1997) and bringing the two promoters in proximal distance ()<br />
additional cellular factors bound to p5 promoter interact with the p19 promoter<br />
leading to transcriptional activation of Rep52/40 (Lackner &amp; Muzyczka 2002). </p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:215.65pt'><br />
<td width=315 valign=top style='width:235.95pt;padding:0cm 5.4pt 0cm 5.4pt;<br />
height:215.65pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><img border=0 width=321 height=250 id="Grafik 1288"<br />
src="Introductionwiki-Dateien/image009.png"<br />
alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Bea\Rep proteins\Freiburg10_SP1_RBE_Interaction_p5_p19.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 7: Forming of the DNA loop brings the p5 rep binding element in proximal distance to<br />
the Sp1 site foud in the p19 promoter.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994052">p40<br />
promoter</a></h5><br />
<br />
<p class=MsoNormal><span lang=EN-GB>The P40 promoter is derived from the<br />
adeno-associated virus serotype 2 (AAV2) genome, where it is located at 40 map<br />
units. It regulates the transcription of the capsid proteins VP1, VP2 and VP3</span><span lang=EN-GB>(Labow, Hermonat, &amp; Berns, 1986</span><span lang=EN-GB>; </span><span lang=EN-GB>Cassinotti, Weitzand, &amp; Tratschin, 1988)</span><span lang=EN-GB>.<br />
</span></p><br />
<br />
<p class=MsoNormal><span lang=EN-GB>Several sequence regions have been<br />
identified to be important for maximal promoter activity: Two Sp1 sites, which<br />
are located 250 (Sp1-50) and 270 (GGT-70) base pairs upstream of the<br />
transcriptional start point and to which Sp1 or Sp1-like proteins bind </span><span lang=EN-GB>(Pereira &amp; Muzyczka 1997)</span><span lang=EN-GB>. </span></p><br />
<br />
<p class=MsoNormal><span lang=EN-GB>Referring to the virus genome, p40 can also<br />
be induced through transactivation by the Rep proteins. The Sp1-50, together<br />
with the CArG-140 site of the P19 promoter, are the main elements involved in<br />
this process. The Rep proteins, which bind to the Rep binding element in the<br />
terminal repeat or the P5 promoter, can induce P19 or P40 by interaction with<br />
their bound Sp1 proteins thereby forming a DNA-loop </span><span<br />
lang=EN-GB>(Pereira &amp; Muzyczka 1997)</span><span lang=EN-GB>. In addition<br />
to that, the TATA box, located at 230, is also required for P40 activity.<br />
Furthermore the ATF-80 and the AP1-40 elements are also important for maximal<br />
promoter induction </span><span<br />
lang=EN-GB>(Pereira &amp; Muzyczka 1997)</span><span lang=EN-GB>.</span></p><br />
<br />
<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994664"></a><a<br />
name="_Toc275994053"></a><a name="_Toc274911366">Integration</a></h3><br />
<br />
<p class=MsoNormal><a name="_Toc274911367">The AAV-2 is the only known<br />
mammalian virus that integrates into a specific location in the human genome in<br />
the presence of Rep78/68: Chromosome 19, 19q13.3-qter. The site of integration<br />
was termed AAVS1. </a></p><br />
<br />
<p class=MsoNormal>The mechanism of Rep-mediated integration into AAVS1 is not<br />
yet completely understood and seems to be imprecise and variable. Deletions or<br />
insertions often occur in the integration process(Schultz &amp; Chamberlain<br />
2008). Linden et al. (1996) proposed a mechanism that is consistent with the<br />
observed patterns in which AAV exists in an integrated form:</p><br />
<br />
<p class=MsoNormal>AAVS1 bears a Rep-binding site (RBS) which is similar to the<br />
RBE in the virus genomes ITR. The Rep proteins are able to simultaneously bind<br />
to AAVS1 and the viral RBE, thereby bringing both strands into close proximity<br />
towards each other. After binding to the AAVS1 site, Rep acts as an<br />
endonuclease, the same way it does when binding to the AAV ITR, introducing a<br />
single strand-nick between two thymidine residues close to the binding site. This<br />
produces a free 3’-OH end which acts as a primer for the host cells’ DNA<br />
polymerase. After replicating the displaced strand, the polymerase switches<br />
templates and replicates the AAV DNA, thereby linking AAVS1 and AAV together.<br />
Prior to integration, the AAV genome often exists in circular and/or<br />
concatameric form, resulting in multiple consecutive AAV copies in the host<br />
genome. Another explanation for this phenomenon could be a circularized AAV<br />
monomer that is being replicated several times in a rolling-circle manner<br />
before being integrated into the host genome. </p><br />
<br />
<p class=MsoNormal>Another template switch back to the AAVS1 sequence creates a<br />
second link between virus and host. This integration mechanism leaves single-stranded<br />
gaps that need to be repaired by cellular enzymes before integration is<br />
complete. Since successful integration of AAV depends on these cellular repair<br />
mechanisms, integration happens more frequently in dividing cells, in which<br />
repair functions are more active.</p><br />
<br />
<p class=MsoNormal>Recently, a sequence in the p5 promoter region that enhances<br />
site-specific integration through interaction with Rep78/68 has been identified,<br />
this motif was labeled p5 integration enhancer element (p5IEE). Apparently, p5IEE<br />
is sufficient to create the AAVS1 - Rep68/78 - Viral DNA -complex necessary for<br />
specific integration, even if the ITRs containing RBEs are not present.</p><br />
<br />
<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994665"></a><a<br />
name="_Toc275994054">Rescue</a></h3><br />
<br />
<p class=MsoNormal>If the latently infected host cell is superinfected with<br />
adenovirus, the integrated virus genome can be rescued from the human<br />
chromosome and proceed its lytic lifecycle. Adenovirus gene products act as<br />
activators on AAV gene expression, leading to an excision of the viral<br />
sequences. Like in the integration process, the Rep 78/68 proteins catalyze the<br />
excision by introduction of single strand nicks at the terminal resolution<br />
sites within the terminal repeat structures flanking the AAV genome. DNA<br />
polymerase, displacing the single-stranded AAV sequence, then elongates the<br />
resulting free 3’ OH ends. The incomplete single-stranded AAV sequence missing<br />
one terminal repeat primes upon itself at the homologous D motives, allowing<br />
DNA polymerase to copy it. This results in full-length, single-stranded AAV molecules,<br />
which are being able to re-enter the replicative cycle (Srivastava 2008), (Samulski 1993).</p><br />
<br />
<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994666"></a><a<br />
name="_Toc275994055">Rep proteins</a><a name="_Toc274911368"></a></h3><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994056">Overview</a></h4><br />
<br />
<p class=MsoNormal>The Adeno-associated virus (AAV) consists of two open<br />
reading frames (ORF), <i>rep</i> and <i>cap</i> ORF. The<i> </i>four<br />
non-structural <i>rep</i> genes are driven by two promoters located at map<br />
units 5 (p5 promoter) and 19 (p19 promoter). Rep proteins are involved in genome encapsidation, regulation of gene expression and replication of the viral genome. </p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:232.5pt'><br />
<td width=533 valign=top style='width:399.65pt;padding:0cm 5.4pt 0cm 5.4pt;<br />
height:232.5pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid;text-autospace:none'><img border=0 width=602 height=225 id="Grafik 37"<br />
src="Introductionwiki-Dateien/image010.png"<br />
alt="Beschreibung: Rep proteins.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>&nbsp;</p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 8: Genomic organization of the AAV-2 genome. The <i>rep</i> gene codes for four<br />
non-structural proteins – Rep40, Rep52, Rep68 and Rep78 – which are involved<br />
in gene regulation, genome encapsidation and viral DNA integration.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>The two larger proteins Rep78/68 play an essential role in<br />
viral genome integration and regulation of AAV gene expression, whereas the<br />
smaller Rep proteins are involved in viral genome encapsidation. Rep proteins<br />
act both as repressors and activators of AAV transcription in respect to the<br />
absence and presence of helper viruses such as adenoviruses (Ad) or herpes<br />
simplex viruses (HSV) by interacting with several cellular proteins (Nash et al. 2009).</p><br />
<br />
<p class=MsoNormal>Furthermore, in the absence of Rep proteins, as it is the<br />
case in recombinant AAVs, integration of the viral genome into the human genome<br />
is rare and random. There are several hotspots for integration of wtAAV genomes<br />
such as the human chromosome 19q13.42, known as the AAVSI site, but as well some<br />
other accessible chromatin regions for preferred integration have been found (5p13.3<br />
and 3p24.3). Integration into the human genome is mediated by the two<br />
regulatory proteins Rep68 and Rep78 driven by the AAV p5 promoter. The proteins<br />
bind to the Rep binding site (RBS) which is located within the inverted terminal<br />
repeats (ITRs). The minimal consensus Rep binding site (RBS) <span<br />
style='font-family:"Courier New"'>GAGT GAGC</span> is found within the ITRs and<br />
in the p5 integration-efficient element (p5IEE) of the p5 promoter (Hüser et al., 2010). Rep78/68 proteins possess DNA-binding (reference), helicase<br />
(reference) and site-specific endonuclease activity located within the first<br />
200 amino acids (Davis et al. 2000). Since the N-terminal region is unique to<br />
the larger Rep proteins, the two smaller Rep proteins possess other biological<br />
functions. Rep52/40 gene expression is driven by the p19 promoter which is<br />
located within <i>rep</i> ORF and the proteins are involved in encapsidating<br />
the viral genome into the preformed capsids. Gene expression of these proteins<br />
is suppressed in absence of adenovirus infection by binding of Rep78/68 to the<br />
p5 promoter. Gene expression of p19 and p40 is transacvtivated by the Rep proteins<br />
Rep78/68 during coinfection.</p><br />
<br />
<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994057"></a><a<br />
name="_Ref275633722"></a><a name="_Toc274911369">Rep 78</a><span<br />
style='font-family:"Dutch801BT-Bold","serif"'> </span></h5><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0<br />
style='border-collapse:collapse;border:none'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Rep78 in a<br />
nutshell:</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• 78 kDa</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• Endonuclease<br />
activity </p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• ATPase<br />
and helicase activity </p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• Regulate<br />
viral gene expression</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• Involved<br />
in genome integration into human chromosome</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>Regulated by the p5 promoter, Rep78 is the largest<br />
non-structural protein found in the wtAAV. Besides regulation of gene<br />
expression and viral genome replication, Rep78 has been found to play a<br />
functional role in AAV site-specific integration into the human genome (Hüser et al. 2010). In absence of Ad helper viruses, overexpression of Rep78 leads to<br />
cell cycle arrest by interacting with cell-cycle regulating phosphatases<br />
causing DNA damage by its intrinsic endonuclease activity (Berthet et al. 2005) and induces apoptosis. Due to its ability to bind to the Rep binding<br />
site (RBS) in the p5 integration-efficient element (p5IEE) of the p5 promoter, Rep78<br />
mediates gene expression and retain a constant level of Rep proteins by<br />
suppressing transcriptional activity of the p5 promoter in absence of Ad<br />
viruses (Yue et al. 2010). Interaction of Rep78 with cellular factors such as<br />
transcription factors (Lackner &amp; Muzyczka 2002) provides the basis for gene<br />
regulation by Rep78 in associated with endogenous molecules. </p><br />
<br />
<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994058"></a><a<br />
name="_Ref275633727"></a><a name="_Toc274911370">Rep 68</a><u> </u></h5><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0<br />
style='border-collapse:collapse;border:none'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Rep68 in a<br />
nutshell</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• 68 kDa</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• Endonuclease<br />
activity </p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• ATPase<br />
and helicase activity </p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• Regulate<br />
gene expression</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• Involved<br />
in genome integration into human chromosome</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>Rep68 is a regulatory protein driven by the p5 promoter with<br />
an apparent molecular weight of 68 kDa lacking 92 amino acids from the carboxy<br />
terminus due to splicing of mRNA coding for the two larger Rep proteins.</p><br />
<br />
<p class=MsoNormal>The non-structural protein Rep68 belongs to the superfamily<br />
3 (SF3) helicase found in other small DNA and RNA viruses such as simian virus<br />
40 (SV40) and bovine papillomavirus (Mansilla-Soto et al. 2009). Formation of oligomeric complexes of Rep proteins provides<br />
the basis for the functional versatility of the two larger regulatory proteins.<br />
The AAA<sup>+</sup> motor domain is known to function as an initiator for<br />
oligomerization of the Rep proteins. The cooperative effect of both domains<br />
appears to be further regulated by ATP binding as well as different DNA<br />
substrates such as dsDNA and ssDNA. Assembly of different nucleoprotein<br />
structures suggest that viral replication and genome integration is regulated<br />
and controlled by distinct Rep complexes which means that in presence of dsDNA<br />
Rep68 assembles to smaller complexes than in presence of ssDNA resulting in<br />
octamers. </p><br />
<br />
<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994059"></a><a<br />
name="_Toc274911372">Rep52</a></h5><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0<br />
style='border-collapse:collapse;border:none'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Rep52 in a<br />
nutshell</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• 52 kDa</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• ATPase<br />
and helicase activity </p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• Involved<br />
in genome encapsidation</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>Rep 52 is under the control of the p19 promoter and shares<br />
the same N-terminus with Rep78. It was shown that Rep52 possesses helicase and<br />
ATPase activity with 3´-5´polarity (Smith &amp; Kotin 1998). Despite the helicase activity, Rep52 and Rep78 share a putative<br />
zinc-finger domain, which suggest interactions with diverse cellular factors (Nash et al. 2009) such as transcription factors (Lackner &amp; Muzyczka 2002) and TATA-binding proteins (Hermonat et al. 1998).</p><br />
<br />
<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994060"></a><a<br />
name="_Toc274911371">Rep40</a></h5><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0<br />
style='border-collapse:collapse;border:none'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Rep40 in a<br />
nutshell</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• 40 kDa</p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• ATPase<br />
and helicase activity </p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>• Involved<br />
in genome encapsidation</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>The smallest Rep protein (Rep40) possesses helicase and<br />
ATPase activity as well, but does not have strict requirements for DNA duplexes<br />
containing a 3´single-stranded end. Rep40 helicase activity requires bivalent<br />
ions such as Mg<sup>2+</sup> or Mn<sup>2+</sup> and is most active using ATP as<br />
substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires<br />
dimerization for functional helicase activity (Collaco et al. 2003). Rep40/52 proteins are required for translocation of the<br />
single-stranded, viral genomes into the preformed capsids proceeding with the<br />
3´end of the DNA (King et al. 2001). </p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:192.55pt'><br />
<td width=285 valign=top style='width:214.0pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt;height:192.55pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'><img border=0<br />
width=270 height=232 id="Picture 8"<br />
src="Introductionwiki-Dateien/image011.jpg"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 9: Crystal structure of the SF-3 helicase (PDB: 1SH9).</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal style='page-break-after:avoid'>&nbsp;</p><br />
<br />
<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994667"></a><a<br />
name="_Toc275994061"></a><a name="_Toc274911373">VP proteins</a></h3><br />
<br />
<p class=MsoNormal>The AAV capsid consists of 60 capsid protein subunits<br />
composed of the three cap proteins VP1, VP2, and VP3, which are encoded in an<br />
overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they<br />
form an icosahedral symmetry. The mRNA encoding for the cap proteins is<br />
transcribed from p40 and alternative spliced to minor and major products.<br />
Alternative splicing and translation initiation of VP2 at a nonconventional ACG<br />
initiation codon promote the expression of the VP proteins. VP1, VP2 and VP3 share<br />
a common C terminus and stop codon, but begin with a different start codon. The<br />
N termini of VP1 and VP2 play important roles in infection and contain motifs<br />
that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear<br />
localization signals (NLSs). These elements are conserved in almost all<br />
parvoviruses. (Johnson et al. 2010a)</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt'>&nbsp;</p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
width=621 style='width:466.1pt;border-collapse:collapse;border:none;<br />
margin-left:-2.25pt;margin-right:-2.25pt'><br />
<tr><br />
<td width=621 valign=top style='width:466.1pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:134.65pt'><br />
<td width=588 valign=top style='width:440.85pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt;height:134.65pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><span style='font-size:12.0pt;line-height:150%'><img border=0<br />
width=574 height=179 id="Grafik 39"<br />
src="Introductionwiki-Dateien/image012.png"<br />
alt="Beschreibung: Cap proteins.png"></span></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 10 Genomic organization of the AAV 2 genome. The <i>cap</i> gene codes for the three<br />
capsid proteins VP1, Vp2 and VP3, which are responsible for the assembly<br />
of the capsid </p><br />
</td><br />
</tr><br />
</table><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994062"></a><a<br />
name="_Toc274911374">VP1</a></h4><br />
<br />
<p class=MsoNormal>Whereas VP1 is translated from the minor spliced mRNA, VP2<br />
and VP3 are translated from the major spliced mRNA. The minor spliced product<br />
is approximately 10-fold less abundant than the major spliced mRNA. Thus, there<br />
is much less VP1 than VP2 and VP3 resulting in a capsid stoichiometric ratio of<br />
1:1:10. The N terminus of VP1 has an extension of 65 amino acids including an<br />
additional extension of 138 N-terminal amino acids forming the unique portion<br />
of VP1. It contains a motif of about 70 amino acids that is highly homologous<br />
to a phospholipase A2 (PLA2) domain. Furthermore, there are nuclear<br />
localization sequences (BR)(+) which are supposed to be necessary for endosomal<br />
escape and nuclearentry. <span lang=DE>(Bleker et al. 2006)</span><span<br />
lang=DE>, </span><span<br />
lang=DE>(Johnson et al. 2010b)</span><span lang=DE>, </span><span<br />
lang=DE>(DiPrimio et al. 2008)</span><span lang=DE>.</span></p><br />
<br />
<p class=MsoNormal><span lang=DE>&nbsp;</span></p><br />
<br />
<p class=MsoNormal>Phospholipases are enzymes that hydrolyze phospholipids into<br />
fatty acids and other lipophilic substances and can be found in mammalian<br />
tissues but also in insect and snake venom. They are subdivided into four major<br />
classes, termed A, B, C and D distinguished by the type of reaction they<br />
catalyse whereas the position of hydrolysis on the glycerol backbone defines<br />
the class of phospholipase.</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;<br />
text-indent:0cm;page-break-after:avoid'><img border=0 width=502 height=273<br />
id="Picture 2064" src="Introductionwiki-Dateien/image013.jpg"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 11: The schematic depiction of some AAV2 domains. VP1 contains a phospholipase A2<br />
(PLA2) domain and four basic regions (BR1–4) located at the N-terminus of VP1<br />
– VP3. The HSPG binding domain is generated by the basic residues at<br />
positions R484, R487, K532, R585, and R588 which are located near the<br />
C-terminus of theVP proteins. The NGR motif 511–513 forms an integrin <span<br />
style='font-family:Symbol'>a</span>5<span style='font-family:Symbol'>b</span>1<br />
binding domain. Adapted from (Michelfelder &amp; Trepel 2009)</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>It specifically recognizes and hydrolyzes the sn-2 acyl bond<br />
of phospholipids releasing arachidonic acid and lysophospholipids.</p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;<br />
text-indent:0cm;page-break-after:avoid'><img border=0 width=437 height=227<br />
id="Picture 2065" src="Introductionwiki-Dateien/image014.jpg"<br />
alt="Beschreibung: Unbenannt1"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 12: adapted from http://www.biochemtech.uni-halle.de/im/1182353660_397_00_800.jpg</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>The reaction mechanism will be depicted by the following<br />
image:</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'><img border=0<br />
width=604 height=485 id="Picture 2066"<br />
src="Introductionwiki-Dateien/image015.jpg"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 13 adapted from “Interfacial Enzymology: The Secreted Phospholipase A2-Paradigm”</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>There are two possible mechanism but the one on the right<br />
side has a lower transient state. The mechanism of sPLA2 on the right side is<br />
initiated by a His-48/Asp-99/calcium complex within the active site. The sn-2<br />
carbonyl oxygen becomes polarized by the calcium ion while also influencing<br />
catalytic water molecule, w5..Via the bridging second water molecule w6 His-48<br />
improves the nucleophilicity of the catalytic water. According to propositions<br />
two water molecules are needed to bypass the distance between the catalytic<br />
histidine and the ester.. Asp-99 is thought to enhance the basicity of His-48<br />
through hydrogen bonding. Substituting the His-48 with an asparagine maintains<br />
wild-typ activity because the functional group on asparagines can function to lower<br />
the pKa of the bridging water molecule, too (Berg et al. 2001). </p><br />
<br />
<p class=MsoNormal><span style='line-height:150%'>The phospholipase A2 is suggested<br />
to mediate membrane disruption of the vesicular compartment, which would allow<br />
escape of the virion into the cytosol, although this has not been demonstrated<br />
for AAV so far.The propensity of cellular PLA2 to cleave phospholipids is usually<br />
regulated by intracellular Ca<sup>2+</sup> levels as well as phosphorylation of<br />
residues near the catalytic domain of the PLA2. Not surprisingly, the<br />
N-terminus of VP1 contains a GXG binding site and several phosphorylation<br />
sites.</span></p><br />
<br />
<p class=MsoNormal><span style='line-height:150%'>&nbsp;</span></p><br />
<br />
<p class=MsoNormal><span style='line-height:150%'>According to the structural<br />
modeling of VP1 the N-terminus can translocate through the 5-fold axis of<br />
symmetry in the capsid and expose the first 185 residues of VP1 (comment:<br />
experimental data also “</span><span style='line-height:150%'>strongly suggest<br />
that N-termini of VP1 </span>harboring the PLA2 domain can be exposed on the<br />
capsid surface through the pores at the fivefold symmetry axes” (Girod et al. 2002)(Bleker et al. 2005a). It only takes about 19 amino acids to reach<br />
through a phospholipid bilayer so this length would be sufficient for the<br />
presentation of the NLS and di-lysine sequences to the cytosol assuming the<br />
PLA2 domain<span style='line-height:150%'> had penetrated through an endosomal<br />
membrane or Golgi. </span><span<br />
style='font-size:10.0pt;line-height:150%'>(Michelfelder &amp; Trepel 2009)</span></p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>Analysing individual steps in the life cycle of several VP1up<br />
mutants and wtAAV-2 lead to the following conclusions: (i) mutations in VP1up<br />
did not affect DNA packaging or replication but resulted in a strong reduction of<br />
infectivity; (ii) this decrease in virus infectivity correlated with a loss in<br />
pvPLA2 activity; (iii) binding to the cell surface and entry into cells was not<br />
affected in VP1up mutants; (iv) however, these mutants showed obviously reduced<br />
and delayed Rep expression. (Girod et al. 2002) Summarizing these results the pvPLA2 activity is required for a step<br />
in the life cycle of the virus following perinuclear accumulation of virions<br />
but (Girod et al. 2002) before the onset of early gene expression.</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>Maybe future work will uncover whether the PLA2 domain in<br />
AAV performs optimally in a specific vesicular compartment, prefers a specific<br />
phospholipid substrate, operates at multiple cellular membranes such as the<br />
endosome and the nuclear envelope, or if its activity is regulated by cellular<br />
components. </p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994063"></a><a<br />
name="_Toc274911375">VP2</a></h4><br />
<br />
<p class=MsoNormal>The translation of VP2 from the major spliced mRNA is less<br />
efficiently compared to the translation of VP3 because it initiates at a Thr<br />
codon (ACG). VP2 and VP1 have an extension at the N terminus that remains<br />
internal when exposing the capsid to experimental conditions like low pH or<br />
heat. The N terminus of VP1 has an extension of 65 amino acids and similar to<br />
VP1 it has two functional elements: a phospholipase A2 (PLA2) domain and nuclear<br />
localization signals (BR)(+). The exact role of VP2 remains unknown, although<br />
the protein is thought to be nonessential for viral assembly and infectivity (Johnson et al. 2010b), (DiPrimio et al. 2008).</p><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994064"></a><a<br />
name="_Toc274911376">VP</a>3</h4><br />
<br />
<p class=MsoNormal>Contained in VP1 and VP2, VP3 is the primary capsid protein<br />
that determines the surface topology of the AAV capsid. The capsid in turn<br />
dictates antigenicity and tropism. In comparison to the initiation of VP1 and<br />
VP2 the initiation of VP3 is because of a Met codon highly efficient (DiPrimio et al. 2008). </p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:222.55pt'><br />
<td width=589 valign=top style='width:441.8pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt;height:222.55pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><img border=0 width=575 height=288 id="Grafik 40"<br />
src="Introductionwiki-Dateien/image016.png" alt="Beschreibung: VP1_2_3.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 14: Genomic organization of the AAV-2 genome. The three capsid proteins VP1, Vp2 and<br />
VP3 have a similar C terminus. The N termini of VP1 and VP2 contain a<br />
phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994065">Natrual<br />
Tropism and HSPG motif</a></h4><br />
<br />
<p class=MsoNormal style='text-autospace:none'>The primary receptor of AAV-2 is<br />
the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006). Its binding motif consists of five amino-acids located on the<br />
capsid surface (Trepel, Vectors, et al. 2009).</p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0<br />
style='border-collapse:collapse;border:none'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid;text-autospace:none'><img border=0 width=628 height=211 id="Picture 39"<br />
src="Introductionwiki-Dateien/image017.png" alt="Beschreibung: Unbenannt"></p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;text-autospace:<br />
none'>Figure 15: adapted from (Trepel, Vectors, et al. 2009). Schematic depiction of the 5 basic amino acids forming<br />
the HSPG motif: R484/R487, K532, R585/587. Other domains or binding motifs<br />
are not shown in this picture.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal style='text-autospace:none'>&nbsp;</p><br />
<br />
<p class=MsoNormal style='text-autospace:none'>HSPG belongs to the<br />
glycosaminoglycanes as well as heparin and consists of heparan sulfate <span<br />
lang=EN>glycosaminoglycan attached to a</span> core protein and can be found on<br />
every human cell surface. </p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;<br />
text-indent:0cm;page-break-after:avoid;text-autospace:none'><img border=0<br />
width=510 height=193 id="Picture 37"<br />
src="Introductionwiki-Dateien/image018.png" alt="Beschreibung: Unbenannt2"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 16: adapted from (Sinnis et al. 2007) amidosulfated disaccharid sequence in heparin.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal style='text-autospace:none'>&nbsp;</p><br />
<br />
<p class=MsoNormal style='text-autospace:none'>Its acid residues bear negative<br />
charges and are therefore prone to electrostatic interactions with e.g. the<br />
positively charged HSPG binding motif of AAV-2. Other interactions with polar<br />
residues are possible, too.</p><br />
<br />
<p class=MsoNormal style='text-autospace:none'>Regarding AAV-2, two point<br />
mutations in AAV-2 (R585A and R588A) are sufficient to eliminate heparin binding<br />
(Opie et al. 2003). The biobricks with this knockout are annotated with<br />
„HSPG-ko“.</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994066"><span<br />
lang=DE>Assembly-activating protein</span></a></h4><br />
<br />
<p class=MsoNormal>A gene encoding for the assembly-activating protein (AAP)<br />
was recently (in 2010) discovered in the Adeno-associated virus (AAV) serotype<br />
2 genome. Its gene product is conserved among all AAV serotypes, illustrating<br />
its essential role in virus life cycle. Its functions comprise transport of the<br />
viral structural proteins to the nucleolus and involvement in following capsid<br />
assembly.</p><br />
<br />
<p class=MsoNormal>The AAP gene, located in the Cap coding region, is<br />
translated from an alternative open reading frame (ORF) with unconventional<br />
start codon. If modifications need to be introduced in the AAV capsid – for<br />
example for targeting approaches – the AAP has to be taken in account in order<br />
to prevent virus assembly impairment.</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994668"></a><a<br />
name="_Toc275994067">Trafficking</a></h3><br />
<br />
<p class=MsoNormal>For creating efficient AAV2 vectors, precise knowledge of<br />
the events following virus transduction is necessary. The subsequent scheme and<br />
summary is intended to be an introduction into the complex process of virus<br />
transduction. </p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:354.75pt'><br />
<td width=422 valign=top style='width:316.8pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt;height:354.75pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><img border=0 width=456 height=510 id="Grafik 123"<br />
src="Introductionwiki-Dateien/image019.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 17: Adapted from (Hildegard Büning et al. 2008)</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>After several contacts with cellular structures like heperan<br />
sulphate proteoglycan (HSPG) the viral capsid proteins get rearranged.<br />
Clathrin-mediated endocytosis and cellular trafficking into thecell’scenter<br />
follows. After acidification and following endosomal escape, the viral genome<br />
is transferred into the nucleus and replicated (lytic phase) or integrated into<br />
the host genome (latent phase). </p><br />
<br />
<p class=MsoNormal><span style='color:black'>Before entering the cell, the<br />
viral particle has in average 4.4 contacts with the cellular surface.</span><span style='color:black'>(Seisenberger et al. 2001)</span><span style='color:black'><br />
The main receptor of AAV2 is heperan sulfate proteoglycan (HSPG). After contact<br />
with HSPG the capsid structure gets rearranged </span><span<br />
style='color:black'>(Levy et al. 2009)</span><span style='color:black'>. This<br />
is probably essential for interaction with other cofactors, which leads to<br />
endocytosis. The factors respectively co-receptors of the cellular surface are<br />
known to enhance the initial binding affinity of HSPG: Fibroblast growth factor<br />
receptor 1 (FGFR-1), hepatocyte growth factor receptor (HGFR) and laminin<br />
receptor. It is known that AAVs affect both: </span><span lang=DE<br />
style='color:black'>&#945;</span><span style='color:black'>V</span><span<br />
lang=DE style='color:black'>&#946;</span><sub><span style='color:black'>5</span></sub><span<br />
style='color:black'>&nbsp;and </span><span lang=DE style='color:black'>&#945;</span><span<br />
style='color:black'>V</span><span lang=DE style='color:black'>&#946;</span><sub><span<br />
style='color:black'>1</span></sub><span style='color:black'>integrin. The </span><span<br />
lang=DE style='color:black'>&#945;</span><span style='color:black'>V</span><span<br />
lang=DE style='color:black'>&#946;</span><sub><span style='color:black'>1</span></sub><span<br />
style='color:black'>&nbsp;-binding site is an asparagine-glycine-arginine motif<br />
</span><span<br />
style='color:black'>(Asokan et al. 2006)</span><span style='color:black'>.<br />
These integrins interact with intracellular molecules like Rho, Rac and Cdc42<br />
GTPases. Figure 2 depicts the following cascade.</span></p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:232.6pt'><br />
<td width=427 valign=top style='width:320.35pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt;height:232.6pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><img border=0 width=420 height=438 id="Grafik 1044"<br />
src="Introductionwiki-Dateien/image020.png"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 18: Adapted from (Sanlioglu et al. 2000a)</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>The initial contact with HSPG, FGFR-1, HGFR and/or laminin<br />
is followed by an interaction with &#945;V&#946;5 and/or &#945;V&#946;1 which<br />
propably leads to an intracellular activation of enzymes involved in the<br />
rearrangement of cytoskeletal proteins like actin, via PI3K-pathway (Kapeller &amp; Cantley 1994), (Li et al. 1998). In general, the receptor-mediated<br />
endocytosis (RME) is a complex process proteins and co-factors form clathrin<br />
coated pits as shown in Figure 16.</p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:249.0pt'><br />
<td width=469 valign=top style='width:351.6pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt;height:249.0pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'><img border=0 width=484 height=330 id="Grafik 59"<br />
src="Introductionwiki-Dateien/image021.png"<br />
alt="Beschreibung: File:Freiburg10 Receptor-mediated endocytosis by clathrin-coated vesicles scheme.PNG"></p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a<br />
name="_Ref275689978">Figure </a>19:</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>The adaptor proteins (APs) AP1, AP2, AP3 and AP4 are<br />
complexes built of four subunits (Collins et al. 2002), (Asokan et al. 2006). Except for AP2, which requires<br />
GTP-bound-Arf1, the APs are linked via phosphatidylinositol (4,5)-bisphosphate<br />
(PIP2) to the cell membrane (Robinson 2004). APs recognize short cytoplasmatic<br />
motifs like YXX-phi (phi: bulky hydrophobic AA) of transmembrane receptors. In general,<br />
the recognition sites (mu-subunits) in the AP-complexes have to be phosphorylated<br />
by kinases (Ohno et al. 1995).</p><br />
<br />
<p class=MsoNormal>The actual scaffold of the endosome is build by the<br />
triskelion formed clathrins. The rigide backbone of clathrins is formed by<br />
three heavy chains (Ybe et al. 1999) and three light chains are regulating<br />
assembly competence (BRODSKY et al. 1991). After building the clathrin<br />
scaffold, dynamine is responsible for pinching-off the clathrin-coated pits (CCPs)<br />
from the cell’s membrane (Summerford &amp; Samulski 1998) (Sanlioglu et al. 2000b). </p><br />
<br />
<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994068">Endosomal<br />
transport and escape</a></h4><br />
<br />
<p class=MsoNormal>Still there are possible additional entering pathways, for<br />
example knocking down microtubuli and microfilament arrangement does not<br />
prevent transduction completely (Kelley 2008). Currently it is thought that endosomal escape happens in the cytoplasma. After<br />
pinching off, the endosomes move via motor proteins along microtubuli and<br />
microfilaments towards the nuclear area. While trafficking through the cell the<br />
early endosoms getting acidulated (Sonntag et al. 2006).</p><br />
<br />
<p class=MsoNormal><span style='color:black'>Additional entering pathways were<br />
postulated for the virus, for example, it has been shown that proteasomal degradation<br />
via ubiquitination hampers transduction efficiency </span><span style='color:black'>(Douar et al. 2001)</span><span style='color:black'>.</span></p><br />
<br />
<p class=MsoNormal><span style='color:black'>The first viral particles in the<br />
nuclear area can be detected after 15 minutes </span><span<br />
style='color:black'>(Seisenberger et al. 2001)</span><span style='color:black'><br />
and an accumulation of virions takes place after 30 minutes post transfection.</span><span<br />
style='color:black'> </span><span style='color:black'>After arrival, the viral<br />
genomes are transported into the nucleus. It is not entirely clear in which way<br />
the transport is accomplished. The viral particles seem to use different<br />
pathways to enter the nucleus, either via the nuclear pore complexes with their<br />
maximal pore size of 23 nm. In this case, the viral capsid (25 nm diameter) has<br />
to be remodeled. Controversial results were published in the past, detecting<br />
intact viral particles (lu et al., 2000), but according to Lux et al. no intact<br />
capsids were detectable when lower amounts of viral particles were transduced </span><span style='color:black'>(Lux et al. 2005)</span><span style='color:black'>.</span></p><br />
<br />
<p class=MsoNormal><span style='color:black'>Obviously further investigation of<br />
intracellular trafficking is essential for optimizing the AAV2 for medical<br />
applications.</span></p><br />
<br />
<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994669"></a><a<br />
name="_Toc275994069">Helper Genes</a></h2><br />
<br />
<p class=MsoNormal>The AAV Helper-Free System by Stratagene (Waldbronn, Germany)<br />
is a modularized system for the production of infectious recombinant AAV-2<br />
virion not depending on a coinfection with any helper virus. It is put into<br />
practice by the three plasmids pHelper, pAAV-RC, recombinant pAAV vector<br />
containing the gene of interest (GOI) and the recombinant cell line AAV-293. </p><br />
<br />
<p class=MsoNormal>The AAV-2 is a replication-deficient parvovirus, which<br />
originally needs a co-infection of adenovirus or herpes virus for replication. To<br />
realize a functional replication of AAV-2 without a co-infection, the AAV<br />
Helper-Free System allocates the pHelper plasmid and the AAV-293 host cells.<br />
The pHelper plasmid encodes for nearly all of the required adenovirus gene<br />
products for replication (VA, E2a, E4). The AAV-293 host cells express stably<br />
the remaining important replication genes (E1A, E1B). </p><br />
<br />
<p class=MsoNormal>Due to the fact that AAV-2 needs all relevant replication<br />
genes for productive infection and that the important replication-genes are<br />
dispersed, the AAV Helper-Free System describes a saver alternative to retroviral<br />
or adenoviral gene delivery (Stratagene n.d.).</p><br />
<br />
<div><br />
<br />
<table cellspacing=0 cellpadding=0 hspace=0 vspace=0 width=542 align=left><br />
<tr><br />
<td valign=top align=left style='padding-top:0cm;padding-right:7.05pt;<br />
padding-bottom:0cm;padding-left:7.05pt'><br />
<p class=MsoCaption>&nbsp;</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>The AAV-293 host cells contain the E1A and E1B genes. The<br />
E1A gene is the first gene to be expressed during an adenovirus infection. The<br />
E1A gene produces two different mRNAs resulting in two different proteins. The<br />
expressed E1A proteins transactivate and induce transcription of other early<br />
genes (like E2 and E4). In this case, E1A proteins do not bind directly onto<br />
control regions, but interact with other host proteins, which are binding to<br />
those regions (Chang et al. 1989) (Modrow et al. 2003).</p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0<br />
style='border-collapse:collapse;border:none'><br />
<tr style='height:261.85pt'><br />
<td width=235 valign=top style='width:176.25pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt;height:261.85pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'><span lang=DE><img<br />
width=216 height=314 src="Introductionwiki-Dateien/image022.png"></span></p><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Figure 20: Schematic overview of the Helper Free System provided by Stratagene.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>In the AAV-2 infection cycle, E1A proteins stimulate the<br />
expression of the p19 promoter and the p5 promoter, which are required to begin<br />
with the <i>rep</i>-gene transcription of the AAV(Chang et al. 1989) (Tratschin et al. 1984).</p><br />
<br />
<p class=MsoNormal>The E1B region encodes two polypeptides with overlapping<br />
reading frames, the major 21-Mr product and the 55-Mr moiety. It has been shown<br />
that only the 55-Mr polypeptide is required for effective helper function. It<br />
enables along with the E4orf6, a stable accumulation of AAV-specific cytoplasmic<br />
RNA, capsid proteins and DNA replication(Samulski &amp; Shenk 1988). In this context it has to be mentioned, that Stratagene<br />
deleted the E4orf6 out of its kit, because of its oncolytic activity. But it<br />
has been explained, that deletion of E4orf6 has no effect on virion production<br />
efficiency(Clark et al. 1999) . </p><br />
<br />
<p class=MsoNormal>The pHelper plasmid exists of the E2a gene, the E4 gene and<br />
the VA gene as well as a pUC ori and an f1 ori. The E2A gene encodes a 72 kD<br />
protein which is produced early in infection (Modrow et al. 2003). One helper function of E2A is to increase the processivity of<br />
replication. In the presence of E2A protein, short replication products, which<br />
are equivalent to break offs of the elongation strand of the template, are<br />
obviously reduced suggestion that E2A supports full-length replication of short<br />
substrates. In immune-depletions, co-localizations between the E2A, the AAV Rep<br />
protein and the AAV DNA have been shown (Ward et al. 1998) .</p><br />
<br />
<div align=center><br />
<br />
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:<br />
-2.25pt'><br />
<tr style='height:238.95pt'><br />
<td width=462 valign=top style='width:346.8pt;padding:0cm 5.4pt 0cm 5.4pt;<br />
height:238.95pt'><br />
<p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:<br />
avoid'>&nbsp;</p><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><img<br />
width=655 height=258 src="Introductionwiki-Dateien/image023.png"></p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<br />
<p class=MsoNormal>It has been reported that E2A has affections on the AAV<br />
promoter regulation of spliced p5 and p19 as well as unspliced p40. E2A could<br />
also be responsible for the production of the AAV capsid proteins (Carter et al. 1992). To which extend it really takes elementary responsiblitiy for any of<br />
the listed functions is not found out yet.</p><br />
<br />
<p class=MsoNormal>The VAI and VAII genes encode for two RNA-species, with a<br />
high GC-percentage and distinct secondary structure. The VAI RNA, which is<br />
implicated to have a helper function in AAV, usually plays a fundamental role<br />
in adenovirus’ protein expression. There it blocks the phosphorylation of the<br />
initiation factor eIF-2, whereby the amino acid chain at the ribosome breaks<br />
off. (Modrow et al. 2003) </p><br />
<br />
<p class=MsoNormal>The expression of the AAV proteins may also be under the VAI<br />
adenovirus control. VAI may increase the AAV capsid production, but it also may<br />
play a role in RNA metabolism.(West et al. 1987) </p><br />
<br />
<p class=MsoNormal>The E4 gene exists of seven open reading frames. In this<br />
content, the proteins occurring from the gene are named E4-ORF1 up to E4-ORF7.<br />
All proteins are under the control of one promoter and arise from alternative<br />
splicing. The E4ORF6 is implicated to have a helper function in AAV. It<br />
promotes the formation of a dsDNA from the genomic ssDNA of the native virus.</p><br />
<br />
<p class=MsoNormal> </p><br />
<br />
<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994670"></a><a<br />
name="_Toc275994070"></a><a name="_Toc274911377">Recombinant Viruses and Mosaic<br />
Viruses</a></h2><br />
<br />
<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994671"></a><a<br />
name="_Toc275994071"></a><a name="_Toc274911378"><span lang=DE>Gene Therapy</span></a></h2><br />
<br />
<p class=MsoNormal>Treating inherited and acquired diseases such as cancer is<br />
still one of the most challenging fields in today’s biomedical research. Ever<br />
since Sidney Farber published a study in 1949 about several folic acid antagonists,<br />
which prevent tumor progression (FARBER 1949), cancer was treated with<br />
chemotherapy, surgery and radiation (Halperin 2006). Nevertheless, due to side effects caused by systemic applications and<br />
the lack of specificity, new treatments must be found for improved therapeutic<br />
efficacy and enhanced selectivity of the anticancer agents. One promising approach<br />
of treating cancer is suicide gene therapy or gene-directed enzyme prodrug<br />
therapy (GDEPT) including two steps of treatment: Targeted introduction of a<br />
gene encoding for enzymes into tumor cells, followed by the administration of a<br />
non-toxic prodrug which is converted into an anti-cancer metabolite. </p><br />
<br />
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left<br />
style='border-collapse:collapse;border:none;margin-left:4.8pt;margin-right:<br />
4.8pt'><br />
<tr><br />
<td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><img<br />
border=0 width=604 height=281 id="Grafik 1155"<br />
src="Introductionwiki-Dateien/image024.png">Figure 21: Schematic overview of<br />
gene-directed enzyme prodrug therapy (GDEPT). The suicide gene is introduced<br />
into the cancer cells. Administration of the prodrug leads to cell death in<br />
the cells expressing the enzyme, which converts the prodrug into the toxic<br />
product.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>Gene delivery using viral vectors to specifically target<br />
cells gained increasing attention in the last years being efficient in<br />
combination with suicide gene therapy. Several prodrug/enzyme systems have been<br />
reported (Greco &amp; Dachs 2001). Ganciclovir (GCV)/herpes simplex virus<br />
thymidine kinase (HSV-TK), 5-fluorocytosine/cytosine deaminase (CD) and<br />
cyclophosphamide/cytochrome P450 systems have been widely used and their activity<br />
has been demonstrated in several preclinical studies (Greco &amp; Dachs 2001).</p><br />
<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>Using transgenic HSV – thymidine kinase or cytosine<br />
deaminase from <i>E. coli</i> for prodrug activation in tumor therapy several<br />
advantages can be found. Besides efficient killing of targeted tumor cells, neighboring,<br />
non-transduced cells are killed as well, providing an important effect in<br />
treating cancer. The bystander phenomenon was first reported by Moolten (1986) showing that HSV-TK negative cells surrounded by HSV-TK positive cells did not<br />
survive prodrug treatment.</p><br />
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<p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 22: Efficient tumor killing is desired in cancer treatment. Locally administered<br />
prodrugs are converted to toxic metabolites by delivered enzmye in the<br />
infected cells. By passive diffusion, gap junction intercellular<br />
communication or immune-related response, non-transduced tumor cells are<br />
killed as well. </p><br />
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<br />
<p class=MsoNormal>&nbsp;</p><br />
<br />
<p class=MsoNormal>Transfer of toxic molecules between transduced and non-transduced<br />
cells can be achieved either through gap junctions (Yang et al. 1998) (Trepel, Stoneham, et al. 2009), via apoptotic bodies (Freeman et al. 1993) or by diffusion of soluble toxic substances (Huber et al. 1993). </p><br />
<br />
<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc274911379"></a><a<br />
name="_Toc275994672"></a><a name="_Toc275994072">Immune Response</a> </h2><br />
<br />
<p class=MsoNormal>Virus infections cause common human disease, including the<br />
familiar cold, influenza, mumps and measles. They are also associated with<br />
severe diseases, for example with Ebola or Marburg fever, with Hepatitis and<br />
AIDS. The immune system protects us from these infections by creating a barrier<br />
that prevents viruses from entering the body or by detecting and eliminating<br />
them in the corpus. </p><br />
<br />
<p class=MsoNormal>Every virus lives and reproduces in its own specific host.<br />
Reproduction can only take place in cell cytoplasm, whose components are needed<br />
in order to compensate for the lack of virus metabolism and<br />
biosynthesis-appliance. Viruses, which are located outside the cells, can be<br />
detected by antibodies, triggering an immune response by the members of the<br />
innate immune system, such as macrophages, neutrophils and natural killer-cells.</p><br />
<br />
<p class=MsoNormal>Inside a cell the virus can only be detected by cytotoxic T<br />
cells: While it uses the cellular machinery for reproduction, some of the viral<br />
proteins are degraded by proteasomes and become presented on the cell surface<br />
by MHC-I-peptides. These exposed virus components are recognized by cytotoxic<br />
CD8+ T cells, which induce death of infected cells. The degraded viral proteins<br />
can also be accessible on MHC-II-peptides, which are detected by CD4+ T helper<br />
cells, which trigger and enforce the immune response for example by production<br />
of specific antibodies. </p><br />
<br />
<p class=MsoNormal>The Adeno-associated virus (AAV) is not associated with any<br />
human disease. Nevertheless, usage of recombinant AAV (rAAV) as therapeutic<br />
vector system harbors risks of immune responses. </p><br />
<br />
<p class=MsoNormal>AAV establishes a latent infection and often integrates at a<br />
specific site on q arm of chromosome 19, which is termed AAVSI site (Hernandez<br />
et al., 1999). This leads to several obstacles for usage of AAV vectors for<br />
therapeutic applications like gene silencing, insertions in gene sequences and<br />
immunotoxocity, a dangerous immune response to the vector or the transgene product<br />
(Mingozzi &amp; High 2007). Humans are the only natural hosts for AAV-2 besides<br />
rhesus macaques. Due to wild-type AAV infections humans keep a population of<br />
antigen-specific memory CD8+ T cells (Mingozzi &amp; High 2007). IgG antibodies<br />
are predominantly involved in the secondary immune response. 91% of Irish blood<br />
donors show a high repertoire of specific IgG1 and IgG2 subclasses and low<br />
doses of IgG3 (Madsen et al. 2009). </p><br />
<br />
<p class=MsoNormal>In vivo studies with AAVlacZ show that AAV vectors induce<br />
the secretion of chemokines and cytokines like gamma interferon (IFN-&#947;)<br />
(Zaiss et al. 2002). Studies in vitro show responses of IFN-&#947;, interleukin<br />
10 (IL-10) and interleukin 13 (IL-13) after stimulation peripheral blood<br />
mononuclear cells (PBMC) from donors with AAV-2. This demonstrates a reaction<br />
of long-live CD4+ T helper-cells that are reactivated (Madsen et al. 2009). These<br />
results reveal that most Europeans are already infected with wildtype-AAV-2.<br />
Researchers suggest that more than 30% of mankind is already infected. In vitro<br />
studies from the United States support this hypothesis. One group found<br />
anti-AAV-antibodies in the blood sera at 80% of randomly chosen volunteers<br />
(Moskalenko et al. 2000). Other investigators show that 0,14% of the examined<br />
CD8+ T cells purified from PBMC are capsid specific for AAV-2 (Mingozzi, Maus,<br />
et al. 2007). These preexisting memory-CD8+ T cells could be responsible for<br />
the difference in vector-infusion outcome between humans (the natural host) and<br />
other species. </p><br />
<br />
<p class=MsoNormal>AAV-2 use distinct cellular receptors, e.g. heparin sulfate<br />
proteoglycan (HSPG), &#945;V&#946;5 integrin and human fibroblast growth factor<br />
receptor 1 (FGFR1) to become internalized (Favaro et al. 2009). These findings<br />
led researchers to the conclusion that the presence of an intact heparinbinding<br />
motif and the capsid t-cell responses are correlated. One group ablated the<br />
heparin-binding site in AAV-2 and observed no CD8+ T cell response. But it did<br />
not seem to influence T helper responses as measured by IgG isotypes and<br />
antigen-stimulated secretion of cytokines (Vandenberghe et al. 2006).</p><br />
<br />
<p class=MsoNormal>Approaches using peptides derived from the sequence of the<br />
VP1 viral capsid protein revealed a total of 59 t-cell epitopes. This<br />
demonstrates the difficulty to avoid the immune system by modifying the AAV<br />
capsid (Madsen et al. 2009). Other approaches in mice reveal that different<br />
serotypes of AAV show the ability to cross-react with existing memory-T cells<br />
(Sabatino et al. 2005). Also in dogs different AAVs use some common peptides on<br />
their surface to activate the immune system (Wang et al. 2010). This shows the<br />
high conservation of the epitopes among multiple serotypes of AAV. </p><br />
<br />
<p class=MsoNormal>While proposing several possible solutions to avoid the<br />
immune system, the polymorphic nature of the human MHC and the high<br />
conservation of peptides on the surface of different serotypes of AAV may<br />
complicate these approaches (Mingozzi, Hasbrouck, et al. 2007). In general it<br />
can be said that the immune response to AAV is not severe as caused by other<br />
virus-types. This is due to the fact that AAVs fail to trigger inflammatory<br />
reactions dendritic cells need to differentiate into professional<br />
antigen-presenting cells (Mingozzi, Maus, et al. 2007). These<br />
antigen-presenting cells are needed for the activation of CD4+ T helper-cells<br />
which are needed for the completely feedback to the immune system. Nevertheless<br />
dendritic cells can be activated through the ability of AAV-2 to bind the HSPG<br />
binding motif with resultant AAV2 antigen inclusion, processing and MHC-I<br />
presentation (Wang et al. 2010). CD4+ T helper-cells can also be activated by other<br />
antigen-presenting cells therefore it is conceivable to block CD4+ cells during<br />
treatment with AAV. The activation of CD8+-t-cells through CD4+ T cells is<br />
depleted and the immune response is even more reduced than within the normal<br />
infection process. </p><br />
<br />
<p class=MsoNormal>Some researchers have found AAV vector DNA in the semen of<br />
dogs and fear the risk of germline transmission (Jiang et al. 2006) although<br />
these findings are controversially discussed. In a rabbit model it was<br />
demonstrated that semen was just positive for vector sequences following<br />
intravascular injection but not following intramuscular injection. Infectious<br />
vector particles were just detected up to four days after treatment and were<br />
undetectable thereafter. So the investigators suggest that AAV-2 presents a low<br />
risk of germline transmission for humans and there is no contemplation for male<br />
infertility so far (Favaro et al. 2009). </p><br />
<br />
<p class=MsoNormal>AAV vectors have been used in several phases of clinical<br />
trials for Leber’s congenital amaurosis (LCA), hemophilia B, Cystic fibrosis,<br />
Arthritis, Muscular dystrophy, Parkinson’s disease, Canavan's disease,<br />
Alzheimer's disease, Batten's disease and Hereditary emphysema.</p><br />
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==Layers of specificity==
=Introduction to Adeno-Associated Virus Se...'</p>
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=Overview=<br />
==The Experimental System==<br />
==Layers of specificity==<br />
=Introduction to Adeno-Associated Virus Serotype 2==<br />
==Biology of the AAV-2==<br />
===Genomic organization===<br />
====Organization of the Inverted Terminal Repeat Structure====<br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/Project_DescriptionTeam:Freiburg Bioware/Project/Project Description2010-10-27T23:55:40Z<p>Achim: </p>
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Therapy using viral vectors is an promising approach for....<br />
In an first step the plasmids of the AAV-2 Helper-free System were genetically modifyed by converting it into BioBricks and inserting of targeting molecules into the constructs. These plasmids were then used to transfect the producer cell line AAV-293. After an incubation of three days the viral vectors were harvested and used to transduce different target cells. The succesful transduction can then for example be measured by detecting the fluorescence of fluorescent proteins in the target cells.<br><br><br />
The majority of the modifications that were introduced into the viral vector aimed to allow differential targeting of tumor cell over healthy off-target cells.<br />
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<center><h2>Layers of specificity</h2></center><br />
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Employment of viral vectors for means of therapy is idea in the context of personalized medicie that gets more and more interest. In such applications the reduction of side effects and the safety of the patient in general is of the highest priority.<br><br />
In order to satisfy this requirement we designed our Therapy Vector with several layers of Specificity:<br><br />
<ul><li>The targeting of the viral vector towards the desired target cell (e.g. tumor cells) is the basic idea behind the emplyment of viral vectors for therapeutical means. There for the natural tropismn has to be knocked down and a desired tropism has to be introduced that allows differential targeting of pathological but not of off-target cells. To fulfill this mission our Virus Construction Kit offers you different solutions.</li><br />
<li>Off-target cells that were transduced by mistake can be preserved from an undesired therapy effect when the therapeutic gene is controley by a tissue specific promoter. For this mean a promoter has to be used that is as specific for the pathological tissue as possible. We included the human telomerase promoter (<a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K404106>phTERT</a>) which is often activated in tumor cells and is there for able to allow differential experssion of a therapeutic geneproduct in pathological cells.</li> <br />
<li>For reasons of safety Therapeutic vector do not directly trigger appoptosis in the successfully targeted cells. To include one further layer of specificity and safety we decided to arm our therapy vector with different prodrug convertases. Neither the single application of the harmless prodrug nor the single expression of the convertase has a noteworthy effect of the transduced cell. Only in cells that express the prodrug convertase and have a sufficient cytoplasmatic concentration of the belonging prodrug apoptosis is triggered. This dependency of the therapy on a prodrug can be employed to protect tissues or other persons that could come in contact with the therapeutical vector. This aspect was specially inportant for the development of a viral vector that is able to infect humans in the context of a undergraduate project for the iGEM competition. Therefor this approach gained our preference over other possibly equivalent arming possibilities described in the tumor therapy with viral vectors.</li></ul><br />
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<center><h2>Modularization of the rAAV genome</h2></center><br />
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<center><h2>Modulatization of the Vectorplasmid</h2></center><br />
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<center><h2>Modification of the viral surface in one cloning step - The ViralBrick standard</h2></center><br />
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<!---Insert text in here---> AAV bears its natural tropism for HSPG on one of its major exposed surface loops - at amino acid position 585/588. Insertions of small binding motives into this region as well as into another surface loop at amino acid position 453 have proved effective for retargeting the virus towards different receptors. <br><br />
We introduced two pairs of single cutting restriction sites into each surface loop, allowing an easy swapping of different loop insertions. Our kit comes with a number of those loop insertion motives, labeled ViralBricks. We do not only provide ViralBricks for differential targeting and knocking out of the natural tropism, we also included inserts for purification and detection of virus particles. <br />
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<center><h2>Specific biotinylation of the Viral Shell - The Biotinylation Acceptor Peptide (BAP)</h2></center><br />
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<!---Insert text in here---> The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
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<center><h2>Purification of Therapeutic Viral Vectors - The His-Affinity Tag</h2></center><br />
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<!---Insert text in here--->Protein tagging via histidine tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are fused to the end of the targeting protein.<br />
The high binding affinity of histidine towards metal is being exploited for the purification of proteins via the so called “Immobilized Metal Ion Affinity Chromatography“(IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein is then applied to a column containing immobilized Ni2+-ions. The His-tags complex the Ni2+-ions while other cellular proteins can be washed off the column. The purified proteins can then be eluted with imidazole, which displaces the histidine residues.(M. C. Smith et al. 1988), (Hoffmann & Roeder 1991) <br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. Koerber et al. have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC (Koerber et al. 2007). For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing for an easy insertion into the 453 and/or 587 loop.<br />
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<center><h2>Targeting of integrin overexpressing cells - The RGD Motif</h2></center><br />
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<!---Insert text in here---> Integrins are transmembrane proteins that, among other functions, mediate cell attachment to surrounding tissues. They bind to a motif consisting of the amino acids arginine, glycine and aspartic acid (RGD in one-letter code). Because Integrin is highly expressed in many tumor cell lines (S. M. Albelda et al. 1990), (Damjanovich et al. 1992), (Lessey et al. 1995), (Smythe et al. 1995), (Gladson & Cheresh 1991), AAV particles displaying the RGD motif on various positions in their capsid proteins have been created by (Shi et al., 2003). Particles displaying RGD at amino acid positions 584 & 588 as well as 453 or 587 (Boucas et al., 2009) showed transduction efficiencies similar to wt AAV, even when the cells’ HSPG receptors were blocked by heparin sulfate or when the natural HSPG binding motif on the capsid surface was knocked out. To further broaden the area of therapeutic application, we created a ViralBrick containing the RGD motive to specifically target cells with low HSPG-/high Integrin expression.<br />
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<center><h2>Arming the Viral Vector with therapeutic antibodies - The Z34C Motif</h2></center><br />
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<!---Insert text in here--->The idea of this targeting approach is to utilize a minimized fragment of the Staphylococcal Protein A that was first described in Staphylococcus aureus. These gram-positive bacteria have evolved the 508 amino acid long protein A that has a high affinity for the Fc-domain of antibodies to protect itself from the immune system. Binding to the constant region of the antibodies is accomplished by the Z-Domain of Protein A that is 58-59 amino acids long, has alone a high affinity (Kd= 14,9 nM) for the antibodies and a three-helix bundle structure. In [Braisted & Wells; 1996] the authors reduced the secundary structure to an two-helix bundle. This size reduction has lead to an drastic reduction of the affinity for IgG (>10^5 fold) which could be recovered by 13 amino acid exchanges resulting in a 38 amino acid long peptide with an satisfying affinity for IgG (Kd = 185 nM) termed Z38. This binding domain was subsequently improved in [Starovasnik et al.; 1997] by the insertion of a disulfide bridge connecting the ends of the helices leading to the binding domain Z34C which shows an increased affinity for IgG (Kd = 20 nM).<br />
<br><br />
This engineered antibody binding domain of 34 amino acids was then inserted into capsids of different viral vectors amongst others also the AAV. In [Ried et al.; 2002] the Z34C domain was inserted at position 587 into the capsid of the AAV resulting in viral vector that can be targeted to different target cells without genetic engineering. This targeting approach was then improved in [Gigout et al.; 2005] by the creation of mosaic vectors that contain only ~25% of recombinant VP-Proteins what resulted in 4 to 5 orders of magnitude more infectiosity compared to all-mutant viruses.<br />
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<center><h2>Testing the limit for loop insertion - The Beta-Lactamase</h2></center><br />
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<center><h2>All-In-One: Testing multiple modified Viral Vectors</h2></center><br />
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<center><h2>Virus Construction Kit - The Manual</h2></center><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:33:33Z<p>Achim: /* Infectious titer */</p>
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<h1>Methods</h1><br />
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<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
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<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
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<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
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<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
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===EM===<br />
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Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
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Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
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===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
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==ITR cloning==<br />
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[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
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As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
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<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
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==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
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===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
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<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
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The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
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===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
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===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
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As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
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===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
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Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
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Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
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For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
<br><br><br><br><br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
*Day one:<br />
**Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
**Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
**Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
**Leave some wells empty for negative control <br />
**Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
*Day two: <br />
**Remove medium (carefully!)<br />
**Treat cells with the drug<br />
**Final volume should be 200 µl per well<br />
**Incubate 1-3 days<br />
*Day three:<br />
**Remove medium (carefully!)<br />
**Fill in 100 µl medium and 25µl MTT solution<br />
**Incubate 4 hours<br />
**Remove medium<br />
**Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
**(take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
The quantitative real-time polymerase chain reaction (qPCR) represents a beneficial method for monitoring the amplification of a specific DNA sequence. The amount of PCR product is measured after each cycle. By comparing the exponential phase of the samples with a standard curve of known sequence copies, the exact initial DNA-amount can be determined. This method not only provides the possibility of precise quantification but also renders analysis of the products after PCR redundant. <br />
For measuring the sequence copy number one takes advantage of a fluorescent dye that incorporates into double-stranded DNA. The template amplification results in increasing fluorescence signals which can be directly correlated to the amplicons. By illustrating the fluorescence against the cycle number by a diagram, quantification of PCR product can be illustrated over time (Nolan et al. 2006; Invitrogen 2008).<br />
<br />
===SYBR Green===<br />
[[Image:Freiburg10_fluor_luoresc.png|thumb|Excitation and Emission Spectra of Sybr Green (adapted from Invitrogen Fluorescence-SpectraViewer; http://tools.invitrogen.com/content/sfs/manuals/cms_039996.pdf)]]<br />
SYBR Green is a nucleic acid stain which preferentially binds within the minor groove of double-stranded DNA. By excitation at λmax = 488 nm, fluorescence emission can be detected at λmax = 525 nm. As binding to double-stranded DNA increases fluorescence emission, amplification of DNA can be monitored and correlated to the amount of double-stranded DNA in the original sample.<br />
<br />
<br><br><br><br><br><br><br><br />
<br />
===Protocols===<br />
We used quantitative real-time PCR to determine genomic virus titers after the transfection of our production cells and after the transduction of our various tumor cell lines with the harvested virus particles. <br />
We followed protocols published by Rohr et al. (Rohr et al. 2002), (Rohr et al. 2005).<br />
====Genomic titer====<br />
[[Image:Freiburg10_genomic_titer.png|thumb|Quantification of virus DNA.]]<br />
To measure the titer of assembled virus particles in our harvested production cells, we first digested all cellular and plasmid DNA left in our virus stocks. We therefore treated 5 µl supernatant from pelleted cell lysate with 7.5 µl DNase I (Fermentas, Catalogue No. EN0521, 1 u/µl) and 5 µl 50 mM MgCl2 (end concentration 5 mM) in a final volume of 50 µl at 37°C for 30 min. We then heat inactivated the enzyme for 10 min at 65°C. PCR reactions were carried out with 2 µl of our digested samples. <br />
====Infectious titer====<br />
[[Image:Freiburg10_infectious_titer.png|thumb|Quantification of DNA in infected cells.]]<br />
To measure the titer of infectious virus particles, we digested our transduced cells with 10µg Proteinase K (Sigma Aldrich) for 1 h at 50°C. After inactivation at 97°C for 15 minutes, we centrifuged the lysate at 13.000 g for 10 minutes and digested 10 µl of the supernatant with 5µl S1 nuclease (Promega, 100 u/µl) for 30 minutes at 37°C. The enzyme was again inactivated for 15 minutes at 97°C. DNA was diluted 1:100. PCR reactions were carried out with 5 µl of our digested samples. <br />
<br />
<br />
All qPCR reactions were carried out using the QuantiFast SYBR Green PCR Kit from Quiagen, (Catalouge No. 204052), employing the following primers:<br />
*CMV_forward_qPCR: 5' - GGGACTTTCCTACTTGGCA - 3'<br />
*CMV_reverse_qPCR: 5' - GGCGGAGTTGTTACGACA - 3'<br />
The qPCR reactions were run on a Corbett RotorGene 3000 realtime thermal cycler and analyzed with the RotorGene software. The qPCR program was:<br />
<br />
[[Image:Freiburg10_PCR_program.png|none|thumb|PCR Program|600px]]<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:29:46Z<p>Achim: /* SYBR Green */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
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<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
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After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
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<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
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[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
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Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
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Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
<br><br><br><br><br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
*Day one:<br />
**Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
**Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
**Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
**Leave some wells empty for negative control <br />
**Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
*Day two: <br />
**Remove medium (carefully!)<br />
**Treat cells with the drug<br />
**Final volume should be 200 µl per well<br />
**Incubate 1-3 days<br />
*Day three:<br />
**Remove medium (carefully!)<br />
**Fill in 100 µl medium and 25µl MTT solution<br />
**Incubate 4 hours<br />
**Remove medium<br />
**Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
**(take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
The quantitative real-time polymerase chain reaction (qPCR) represents a beneficial method for monitoring the amplification of a specific DNA sequence. The amount of PCR product is measured after each cycle. By comparing the exponential phase of the samples with a standard curve of known sequence copies, the exact initial DNA-amount can be determined. This method not only provides the possibility of precise quantification but also renders analysis of the products after PCR redundant. <br />
For measuring the sequence copy number one takes advantage of a fluorescent dye that incorporates into double-stranded DNA. The template amplification results in increasing fluorescence signals which can be directly correlated to the amplicons. By illustrating the fluorescence against the cycle number by a diagram, quantification of PCR product can be illustrated over time (Nolan et al. 2006; Invitrogen 2008).<br />
<br />
===SYBR Green===<br />
[[Image:Freiburg10_fluor_luoresc.png|thumb|Excitation and Emission Spectra of Sybr Green (adapted from Invitrogen Fluorescence-SpectraViewer; http://tools.invitrogen.com/content/sfs/manuals/cms_039996.pdf)]]<br />
SYBR Green is a nucleic acid stain which preferentially binds within the minor groove of double-stranded DNA. By excitation at λmax = 488 nm, fluorescence emission can be detected at λmax = 525 nm. As binding to double-stranded DNA increases fluorescence emission, amplification of DNA can be monitored and correlated to the amount of double-stranded DNA in the original sample.<br />
<br />
<br><br><br><br><br><br><br><br />
<br />
===Protocols===<br />
We used quantitative real-time PCR to determine genomic virus titers after the transfection of our production cells and after the transduction of our various tumor cell lines with the harvested virus particles. <br />
We followed protocols published by Rohr et al. (Rohr et al. 2002), (Rohr et al. 2005).<br />
====Genomic titer====<br />
[[Image:Freiburg10_genomic_titer.png|thumb|Quantification of virus DNA.]]<br />
To measure the titer of assembled virus particles in our harvested production cells, we first digested all cellular and plasmid DNA left in our virus stocks. We therefore treated 5 µl supernatant from pelleted cell lysate with 7.5 µl DNase I (Fermentas, Catalogue No. EN0521, 1 u/µl) and 5 µl 50 mM MgCl2 (end concentration 5 mM) in a final volume of 50 µl at 37°C for 30 min. We then heat inactivated the enzyme for 10 min at 65°C. PCR reactions were carried out with 2 µl of our digested samples. <br />
====Infectious titer====<br />
[[Image:Freiburg10_infectious_titer.png|thumb|Quantification of DNA in infected cells.]]<br />
To measure the titer of infectious virus particles, we digested our transduced cells with 10µg Proteinase K (Sigma Aldrich) for 1 h at 50°C. After inactivation at 97°C for 15 minutes, we centrifuged the lysate at 13.000 g for 10 minutes and digested 10 µl of the supernatant with 5µl S1 nuclease (Promega, 100 u/µl) for 30 minutes at 37°C. The enzyme was again inactivated for 15 minutes at 97°C. DNA was diluted 1:100. PCR reactions were carried out with 5 µl of our digested samples. <br />
<br />
<br />
All qPCR reactions were carried out using the QuantiFast SYBR Green PCR Kit from Quiagen, (Catalouge No. 204052), employing the following primers:<br />
*CMV_forward_qPCR: 5' - GGGACTTTCCTACTTGGCA - 3'<br />
*CMV_reverse_qPCR: 5' - GGCGGAGTTGTTACGACA - 3'<br />
The qPCR reactions were run on a Corbett RotorGene 3000 realtime thermal cycler and analyzed with the RotorGene software. The qPCR program was:<br />
<br />
[[Image:Freiburg10_PCR_program.png|none|thumb|PCR Program|300px]]<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:28:30Z<p>Achim: /* Quantitative real-time PCR */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
<html><br />
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<tbody><br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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</p><br />
</p><br />
</td><br />
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<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
<p><br />
</p><br />
</p><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</div><br />
</html><br />
===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
<br><br><br><br><br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
*Day one:<br />
**Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
**Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
**Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
**Leave some wells empty for negative control <br />
**Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
*Day two: <br />
**Remove medium (carefully!)<br />
**Treat cells with the drug<br />
**Final volume should be 200 µl per well<br />
**Incubate 1-3 days<br />
*Day three:<br />
**Remove medium (carefully!)<br />
**Fill in 100 µl medium and 25µl MTT solution<br />
**Incubate 4 hours<br />
**Remove medium<br />
**Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
**(take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
The quantitative real-time polymerase chain reaction (qPCR) represents a beneficial method for monitoring the amplification of a specific DNA sequence. The amount of PCR product is measured after each cycle. By comparing the exponential phase of the samples with a standard curve of known sequence copies, the exact initial DNA-amount can be determined. This method not only provides the possibility of precise quantification but also renders analysis of the products after PCR redundant. <br />
For measuring the sequence copy number one takes advantage of a fluorescent dye that incorporates into double-stranded DNA. The template amplification results in increasing fluorescence signals which can be directly correlated to the amplicons. By illustrating the fluorescence against the cycle number by a diagram, quantification of PCR product can be illustrated over time (Nolan et al. 2006; Invitrogen 2008).<br />
<br />
===SYBR Green===<br />
[[Image:Freiburg10_fluor_luoresc.png|thumb|Excitation and Emission Spectra of Sybr Green (adapted from Invitrogen Fluorescence-SpectraViewer; http://tools.invitrogen.com/content/sfs/manuals/cms_039996.pdf)]]<br />
SYBR Green is a nucleic acid stain which preferentially binds within the minor groove of double-stranded DNA. By excitation at λmax = 488 nm, fluorescence emission can be detected at λmax = 525 nm. As binding to double-stranded DNA increases fluorescence emission, amplification of DNA can be monitored and correlated to the amount of double-stranded DNA in the original sample.<br />
<br />
<br><br><br><br><br><br />
===Protocols===<br />
We used quantitative real-time PCR to determine genomic virus titers after the transfection of our production cells and after the transduction of our various tumor cell lines with the harvested virus particles. <br />
We followed protocols published by Rohr et al. (Rohr et al. 2002), (Rohr et al. 2005).<br />
====Genomic titer====<br />
[[Image:Freiburg10_genomic_titer.png|thumb|Quantification of virus DNA.]]<br />
To measure the titer of assembled virus particles in our harvested production cells, we first digested all cellular and plasmid DNA left in our virus stocks. We therefore treated 5 µl supernatant from pelleted cell lysate with 7.5 µl DNase I (Fermentas, Catalogue No. EN0521, 1 u/µl) and 5 µl 50 mM MgCl2 (end concentration 5 mM) in a final volume of 50 µl at 37°C for 30 min. We then heat inactivated the enzyme for 10 min at 65°C. PCR reactions were carried out with 2 µl of our digested samples. <br />
====Infectious titer====<br />
[[Image:Freiburg10_infectious_titer.png|thumb|Quantification of DNA in infected cells.]]<br />
To measure the titer of infectious virus particles, we digested our transduced cells with 10µg Proteinase K (Sigma Aldrich) for 1 h at 50°C. After inactivation at 97°C for 15 minutes, we centrifuged the lysate at 13.000 g for 10 minutes and digested 10 µl of the supernatant with 5µl S1 nuclease (Promega, 100 u/µl) for 30 minutes at 37°C. The enzyme was again inactivated for 15 minutes at 97°C. DNA was diluted 1:100. PCR reactions were carried out with 5 µl of our digested samples. <br />
<br />
<br />
All qPCR reactions were carried out using the QuantiFast SYBR Green PCR Kit from Quiagen, (Catalouge No. 204052), employing the following primers:<br />
*CMV_forward_qPCR: 5' - GGGACTTTCCTACTTGGCA - 3'<br />
*CMV_reverse_qPCR: 5' - GGCGGAGTTGTTACGACA - 3'<br />
The qPCR reactions were run on a Corbett RotorGene 3000 realtime thermal cycler and analyzed with the RotorGene software. The qPCR program was:<br />
<br />
[[Image:Freiburg10_PCR_program.png|none|thumb|PCR Program|300px]]<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:25:37Z<p>Achim: /* Quantitative real-time PCR */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
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<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<td width="489" valign="top"><br />
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<b>genotype</b><br />
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</p><br />
</p><br />
</td><br />
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<tr><br />
<td width="130" valign="top"><br />
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BL21<br />
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</p><br />
</td><br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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</p><br />
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<td width="489" valign="top"><br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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</p><br />
</p><br />
</td><br />
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<td width="130" valign="top"><br />
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XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
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<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
<br><br><br><br><br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
*Day one:<br />
**Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
**Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
**Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
**Leave some wells empty for negative control <br />
**Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
*Day two: <br />
**Remove medium (carefully!)<br />
**Treat cells with the drug<br />
**Final volume should be 200 µl per well<br />
**Incubate 1-3 days<br />
*Day three:<br />
**Remove medium (carefully!)<br />
**Fill in 100 µl medium and 25µl MTT solution<br />
**Incubate 4 hours<br />
**Remove medium<br />
**Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
**(take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
The quantitative real-time polymerase chain reaction (qPCR) represents a beneficial method for monitoring the amplification of a specific DNA sequence. The amount of PCR product is measured after each cycle. By comparing the exponential phase of the samples with a standard curve of known sequence copies, the exact initial DNA-amount can be determined. This method not only provides the possibility of precise quantification but also renders analysis of the products after PCR redundant. <br />
For measuring the sequence copy number one takes advantage of a fluorescent dye that incorporates into double-stranded DNA. The template amplification results in increasing fluorescence signals which can be directly correlated to the amplicons. By illustrating the fluorescence against the cycle number by a diagram, quantification of PCR product can be illustrated over time (Nolan et al. 2006; Invitrogen 2008).<br />
<br />
===SYBR Green===<br />
[[Image:Freiburg10_fluor_luoresc.png|thumb|Excitation and Emission Spectra of Sybr Green (adapted from Invitrogen Fluorescence-SpectraViewer; http://tools.invitrogen.com/content/sfs/manuals/cms_039996.pdf)]]<br />
SYBR Green is a nucleic acid stain which preferentially binds within the minor groove of double-stranded DNA. By excitation at λmax = 488 nm, fluorescence emission can be detected at λmax = 525 nm. As binding to double-stranded DNA increases fluorescence emission, amplification of DNA can be monitored and correlated to the amount of double-stranded DNA in the original sample.<br />
===Protocols===<br />
We used quantitative real-time PCR to determine genomic virus titers after the transfection of our production cells and after the transduction of our various tumor cell lines with the harvested virus particles. <br />
We followed protocols published by Rohr et al. (Rohr et al. 2002), (Rohr et al. 2005).<br />
====Genomic titer====<br />
[[Image:Freiburg10_genomic_titer.png|thumb|Quantification of virus DNA.]]<br />
To measure the titer of assembled virus particles in our harvested production cells, we first digested all cellular and plasmid DNA left in our virus stocks. We therefore treated 5 µl supernatant from pelleted cell lysate with 7.5 µl DNase I (Fermentas, Catalogue No. EN0521, 1 u/µl) and 5 µl 50 mM MgCl2 (end concentration 5 mM) in a final volume of 50 µl at 37°C for 30 min. We then heat inactivated the enzyme for 10 min at 65°C. PCR reactions were carried out with 2 µl of our digested samples. <br />
====Infectious titer====<br />
[[Image:Freiburg10_infectious_titer.png|thumb|Quantification of DNA in infected cells.]]<br />
To measure the titer of infectious virus particles, we digested our transduced cells with 10µg Proteinase K (Sigma Aldrich) for 1 h at 50°C. After inactivation at 97°C for 15 minutes, we centrifuged the lysate at 13.000 g for 10 minutes and digested 10 µl of the supernatant with 5µl S1 nuclease (Promega, 100 u/µl) for 30 minutes at 37°C. The enzyme was again inactivated for 15 minutes at 97°C. DNA was diluted 1:100. PCR reactions were carried out with 5 µl of our digested samples. <br />
<br />
<br />
All qPCR reactions were carried out using the QuantiFast SYBR Green PCR Kit from Quiagen, (Catalouge No. 204052), employing the following primers:<br />
*CMV_forward_qPCR: 5' - GGGACTTTCCTACTTGGCA - 3'<br />
*CMV_reverse_qPCR: 5' - GGCGGAGTTGTTACGACA - 3'<br />
The qPCR reactions were run on a Corbett RotorGene 3000 realtime thermal cycler and analyzed with the RotorGene software. The qPCR program was:<br />
<br />
[[Image:Freiburg10_PCR_program.png|none|thumb|PCR Program]]<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:17:39Z<p>Achim: /* Protocol */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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</p><br />
</p><br />
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<br />
</p><br />
</p><br />
</td><br />
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</tbody><br />
</table><br />
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</p><br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
<br><br><br><br><br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
*Day one:<br />
**Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
**Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
**Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
**Leave some wells empty for negative control <br />
**Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
*Day two: <br />
**Remove medium (carefully!)<br />
**Treat cells with the drug<br />
**Final volume should be 200 µl per well<br />
**Incubate 1-3 days<br />
*Day three:<br />
**Remove medium (carefully!)<br />
**Fill in 100 µl medium and 25µl MTT solution<br />
**Incubate 4 hours<br />
**Remove medium<br />
**Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
**(take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:16:17Z<p>Achim: /* Protocol */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
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<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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</p><br />
</td><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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<td width="130" valign="top"><br />
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XL10-Gold<br />
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</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
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<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
<br><br><br><br><br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
<br />
<br />
*Day one:<br />
**Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
**Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
**Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
**Leave some wells empty for negative control <br />
**Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
*Day two: <br />
**Remove medium (carefully!)<br />
**Treat cells with the drug<br />
**Final volume should be 200 µl per well<br />
**Incubate 1-3 days<br />
*Day three:<br />
**Remove medium (carefully!)<br />
**Fill in 100 µl medium and 25µl MTT solution<br />
**Incubate 4 hours<br />
**Remove medium<br />
**Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
**(take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:14:58Z<p>Achim: /* Overview */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<td width="130" valign="top"><br />
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<b>Cell strain</b><br />
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</p><br />
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</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
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</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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</p><br />
</p><br />
</td><br />
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<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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</table><br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
<br><br><br><br><br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
<br />
Figure 51: MTT assay<br />
<br />
<br />
*Day one:<br />
**Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
**Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
**Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
**Leave some wells empty for negative control <br />
**Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
*Day two: <br />
**Remove medium (carefully!)<br />
**Treat cells with the drug<br />
**Final volume should be 200 µl per well<br />
**Incubate 1-3 days<br />
*Day three:<br />
**Remove medium (carefully!)<br />
**Fill in 100 µl medium and 25µl MTT solution<br />
**Incubate 4 hours<br />
**Remove medium<br />
**Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
**(take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:13:47Z<p>Achim: /* Protocol */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
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Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
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===Resources===<br />
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1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
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==ITR cloning==<br />
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[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
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As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
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<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
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==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
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===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
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<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
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The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
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PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
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Link to NEB: http://www.neb.com<br />
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===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
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As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
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Link to Agilent: http://www.genomics.agilent.com<br />
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===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
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===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
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As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
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===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
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==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
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The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
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Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
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Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
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For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
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=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
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==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
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After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
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[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
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There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
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[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
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The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
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Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
<br />
Figure 51: MTT assay<br />
<br />
<br />
*Day one:<br />
**Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
**Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
**Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
**Leave some wells empty for negative control <br />
**Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
*Day two: <br />
**Remove medium (carefully!)<br />
**Treat cells with the drug<br />
**Final volume should be 200 µl per well<br />
**Incubate 1-3 days<br />
*Day three:<br />
**Remove medium (carefully!)<br />
**Fill in 100 µl medium and 25µl MTT solution<br />
**Incubate 4 hours<br />
**Remove medium<br />
**Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
**(take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:11:54Z<p>Achim: /* Overview */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
<html><br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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</p><br />
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<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
<p><br />
</p><br />
</p><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
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<br />
</p><br />
</p><br />
</div><br />
</html><br />
===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|MTT assay]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
<br />
Figure 51: MTT assay<br />
<br />
<br />
Day one:<br />
• Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
• Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
• Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
• Leave some wells empty for negative control <br />
• Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
Day two: <br />
• Remove medium (carefully!)<br />
• Treat cells with the drug<br />
• Final volume should be 200 µl per well<br />
• Incubate 1-3 days<br />
Day three:<br />
• Remove medium (carefully!)<br />
• Fill in 100 µl medium and 25µl MTT solution<br />
• Incubate 4 hours<br />
• Remove medium<br />
• Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
• (take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T23:10:49Z<p>Achim: /* Overview */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
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<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
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<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
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The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
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<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
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Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
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For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
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After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
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[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
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There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
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[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
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The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
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Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
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Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
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Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
[[Image:Freiburg10_MTT_reaction.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
[[Image:Freiburg10_MTT_method.png|thumb|The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)]]<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
<br />
Figure 51: MTT assay<br />
<br />
<br />
Day one:<br />
• Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
• Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
• Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
• Leave some wells empty for negative control <br />
• Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
Day two: <br />
• Remove medium (carefully!)<br />
• Treat cells with the drug<br />
• Final volume should be 200 µl per well<br />
• Incubate 1-3 days<br />
Day three:<br />
• Remove medium (carefully!)<br />
• Fill in 100 µl medium and 25µl MTT solution<br />
• Incubate 4 hours<br />
• Remove medium<br />
• Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
• (take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:56:23Z<p>Achim: /* Protocol */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<td width="130" valign="top"><br />
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<b>Cell strain</b><br />
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</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
<p><br />
</p><br />
</p><br />
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<p><br />
<br />
</p><br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
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<br />
</p><br />
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</div><br />
</html><br />
===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
Figure 50 The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)<br />
<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
===Protocol===<br />
Sörensen-buffer: 0.1 M Glycine, 0,1 M NaCl, H2O, pH 10.5<br />
MTT-Solution: 3.65 mg/ml in PBS, <br />
The solution should be kept cold (4°C) and in the dark (Schröter 2009)<br />
<br />
<br />
Figure 51: MTT assay<br />
<br />
<br />
Day one:<br />
• Take the T75 Flask, remove Medium, wash the cells with 8 ml PBS detach cells with 1ml Trypsin (about 30 seconds up to 10 minutes incubation time! Check permantly!). Inactivate Trypsin with 10 ml DMEM medium, transfer the cells into a 15 ml falcon. Centrifugate (200 rcf/g for 5 min).<br />
• Remove supernatant, resuspend pellet with 10 ml DMEM. Count cells via Neubauer Cell Chamber.<br />
• Take the 96 well plates and add 5.000-10.000 cells in each well. Fill up to 200µl with DMEM.<br />
• Leave some wells empty for negative control <br />
• Put the plate into the incubator. Incubate over night, to allow cells to attach to the wells<br />
Day two: <br />
• Remove medium (carefully!)<br />
• Treat cells with the drug<br />
• Final volume should be 200 µl per well<br />
• Incubate 1-3 days<br />
Day three:<br />
• Remove medium (carefully!)<br />
• Fill in 100 µl medium and 25µl MTT solution<br />
• Incubate 4 hours<br />
• Remove medium<br />
• Resuspend in 200µl DMSO and 25 µl Sörensen-buffer<br />
• (take on shaker for 15 minutes) read absorbance at 570 nm<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:54:24Z<p>Achim: /* Overview */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
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<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
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<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
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<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
The MTT-assay is a colorimetric assay, which is able to detect cell proliferation, viability and cytotoxity. It is based on the metabolic activity of viable cells.<br />
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) is a yellow tetrazole, which is reduced to purple formazan in the presence of NADH and NADPH. <br />
<br />
Figure 50 The reaction of the dye MTT (yellow) into the purple product formazan (image from wikipedia)<br />
<br />
<br />
Viable, metabolic active cells produce in the respiratory chain the pyridine nucleotide cofactors (NADH, NADPH). NADH and NADPH are basically responsible for cellular reductions and therefore responsible for the cleavage of MTT. (Roche n.d.) Our purpose is to use the MTT-assay as a cytotoxity test, for testing cytotoxity on the tumor cell lines HT1080 and A431. <br />
The MTT-Assay can easily be performed in 96-well plates. This enables a reduction of the amount of culture medium, cells and plasticware. Furthermore, the dye MTT is a bargain. On balance, it is a cheap and simple method to detect viability. <br />
The colorimetric analysis can simply be carried out via spectrometry. In our case, we are using the ELISA-Reader Tecan Sunrise for reading out our 96-well plates.<br />
In comparison to other viability assays, the product formazan of the MTT-assay unfortunately is water insoluble. That’s why an additionally step to solve the formazan has to be performed.<br />
<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:54:03Z<p>Achim: /* Cell Staining for Flow Cytometry */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<td width="130" valign="top"><br />
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<b>Cell strain</b><br />
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</p><br />
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</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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</p><br />
</p><br />
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<br />
</p><br />
</p><br />
</td><br />
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</table><br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
*7-AAD Viability Staining: 7-AAD has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis.<br />
*For dead cell exclusion, wash cells threefold with 500 µl of 1x Dulbecco`s PBS<br />
*Discard supernatant and resuspend cell pellet in 300 µl of Cell Staining Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Add 3 µl of 7-AAD and incubate for 5-10 minutes in the dark before analysis<br />
<br />
*Alexa Flour 647 Annexin V: Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. <br />
*Wash cells twice with cold cell staining buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany)<br />
*Resuspend cell pellet in 100 µl Annexin V binding buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 5 µl of Alexa Fluor 647 Annexin V (BioLegend, BIOZOL Diagnostica, Eching, Germany) <br />
*Add 10 µl of PI solution (BioLegend, BIOZOL Diagnostica, Eching, Germany) or 7-AAD (for double-staining)<br />
*Gently vortex the cells and incubate for 15 min at RT in the dark<br />
*Add 400 µl of Annexin V Binding Buffer (BioLegend, BIOZOL Diagnostica, Eching, Germany) and analyze the samples by flow cytometry<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:52:07Z<p>Achim: /* Sample Preparation for Flow Cytometry */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
<html><br />
<div><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
<p><br />
</p><br />
<table border="1" cellspacing="0" cellpadding="0"><br />
<tbody><br />
<tr><br />
<td width="619" colspan="2" valign="top"><br />
<p><br />
Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
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</p><br />
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</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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</p><br />
</p><br />
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</p><br />
</p><br />
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</p><br />
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</div><br />
</html><br />
===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
*Harvest cells by trypsinization with 0,25% 1x Trypsin-EDTA (Invitrogen, Darmstadt, Germany) <br />
*Collect cells by centrifugation at 1200 x g for 3 minutes (Heraeus Sepatech, Varifuge 3.0 R, Thermo Scientific, Germany)<br />
*Discard supernatant and wash cells by resuspending cell pellet with 500 µl 1x Dulbecco`s PBS without calcium, and magnesium (PAA, Pasching, Austria)<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes<br />
*Discard supernatant and resuspend cell pellet with 500 µl Dulbecco`s PBS<br />
*Centrifuge at 1200 x g for 3 minutes <br />
*Discard supernatant and resuspend cell pellet with 300 µl Dulbecco`s PBS for evaluation on flow cytometry<br />
*Use the 488 nm blue laser to excite the fluorochrome YFP and FL-1 to detect light emitted.<br />
<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:48:05Z<p>Achim: /* Overview */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
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[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
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<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
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==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
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<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
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The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
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Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
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<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
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For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
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After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
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[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
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There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
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[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
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The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
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Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
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Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:46:41Z<p>Achim: /* Overview */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<td width="130" valign="top"><br />
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<b>Cell strain</b><br />
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</p><br />
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</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
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</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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</p><br />
</p><br />
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<br />
</p><br />
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</td><br />
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</tbody><br />
</table><br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
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<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
<gallery align="center"><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:45:08Z<p>Achim: /* Overview */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
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<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
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<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
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<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
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<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
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<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<td width="130" valign="top"><br />
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<b>Cell strain</b><br />
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</p><br />
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</td><br />
<td width="489" valign="top"><br />
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<b>genotype</b><br />
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</p><br />
</p><br />
</td><br />
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<td width="130" valign="top"><br />
<p><br />
BL21<br />
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</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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</table><br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br />
<br />
<gallery><br />
Image:Freibur10_gating_for_excluding_cell_debris.png|Gating for excluding cell debris<br />
<br />
Image:Freiburg10_histogram_negative_control.png|Histogram of negative control cells<br />
<br />
Image:Freiburg10_histogram_negative_control_II.png|YFP-positive transduced cells<br />
</gallery><br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:43:12Z<p>Achim: /* Overview */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br><br><br><br><br><br><br><br />
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[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
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<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br><br> <br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br><br />
<br />
<br />
[[Image:Freibur10_gating_for_excluding_cell_debris.png|thumb|250px|: Gating for excluding cell debris]]<br />
<br />
[[Image:Freiburg10_histogram_negative_control.png|thumb|250px|Histogram of negative control cells]]<br />
<br />
[[Image:Freiburg10_histogram_negative_control_II.png|thumb|250px|YFP-positive transduced cells ]]<br />
<br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:42:21Z<p>Achim: /* Overview */</p>
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<h1>Methods</h1><br />
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<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
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<br />
===EM===<br />
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<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
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<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
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<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
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<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
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<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles.<br />
<br />
<br><br><br><br> <br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
<br><br><br><br><br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br><br><br><br><br />
<br />
<br />
[[Image:Freibur10_gating_for_excluding_cell_debris.png|thumb|250px|: Gating for excluding cell debris]]<br />
<br />
[[Image:Freiburg10_histogram_negative_control.png|thumb|250px|Histogram of negative control cells]]<br />
<br />
[[Image:Freiburg10_histogram_negative_control_II.png|thumb|250px|YFP-positive transduced cells ]]<br />
<br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:40:56Z<p>Achim: /* Overview */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<td width="489" valign="top"><br />
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<b>genotype</b><br />
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<td width="130" valign="top"><br />
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BL21<br />
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</td><br />
<td width="489" valign="top"><br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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</p><br />
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</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles. <br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br />
[[Image:Freibur10_gating_for_excluding_cell_debris.png|thumb|250px|: Gating for excluding cell debris]]<br />
<br />
[[Image:Freiburg10_histogram_negative_control.png|thumb|250px|Histogram of negative control cells]]<br />
<br />
[[Image:Freiburg10_histogram_negative_control_II.png|thumb|250px|YFP-positive transduced cells ]]<br />
<br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:38:47Z<p>Achim: /* Overview */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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</p><br />
</p><br />
</td><br />
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<tr><br />
<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|right|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles. <br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|right|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|right|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br />
[[Image:Freibur10_gating_for_excluding_cell_debris.png|thumb|right|250px|: Gating for excluding cell debris]]<br />
<br />
[[Image:Freiburg10_histogram_negative_control.png|thumb|right|250px|Histogram of negative control cells]]<br />
<br />
[[Image:Freiburg10_histogram_negative_control_II.png|thumb|right|250px|YFP-positive transduced cells ]]<br />
<br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:28:11Z<p>Achim: /* Overview */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
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<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
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The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
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<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
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For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|right|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
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[[Image:Freiburg10_light-scattering.png|thumb|right|250px|Light-scattering properties of a cell adapted from (Marti, Stetler-Stevenson, Bleesing, & Fleisher, 2001)]]<br />
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After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles. <br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|right|250px|: Excitation/emission spectra of GFP and YFP adapted from (Lybarger et al. 1998)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
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[[Image:Freiburg10_Laser_light_source.png|thumb|right|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
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Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
[[Image:Freibur10_gating_for_excluding_cell_debris.png|thumb|right|250px|: Gating for excluding cell debris]]<br />
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[[Image:Freiburg10_histogram_negative_control.png|thumb|right|250px|Histogram of negative control cells]]<br />
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[[Image:Freiburg10_histogram_negative_control_II.png|thumb|right|250px|YFP-positive transduced cells ]]<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:23:43Z<p>Achim: /* Overview */</p>
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<h1>Methods</h1><br />
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<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
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<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
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<br />
===EM===<br />
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<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
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<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
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<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
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<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
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<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|right|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|right|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles. <br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|right|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
[[Image:Freiburg10_Laser_light_source.png|thumb|right|250px|Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).]]<br />
<br />
<br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:21:58Z<p>Achim: /* Overview */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<td width="130" valign="top"><br />
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<b>Cell strain</b><br />
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</p><br />
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</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
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<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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</p><br />
</p><br />
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<br />
</p><br />
</p><br />
</td><br />
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</tbody><br />
</table><br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
[[Image:Freiburg10_Schematic_overview_flow_cytometer.png|thumb|right|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. <br />
<br />
<br />
[[Image:Freiburg10_light-scattering.png|thumb|right|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
<br />
<br />
After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles. <br />
<br />
[[Image:Freiburg10_Excitation_emission_spectra.png|thumb|right|250px|Schematic overview of a typical flow cytometer setup (Beckmann Coulter 2008)]]<br />
<br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br />
Figure 47: Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).<br />
<br />
<br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:15:18Z<p>Achim: /* Flow Cytometry */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
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<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
<p><br />
</p><br />
</p><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</div><br />
</html><br />
===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
Flow cytometry is a technique for measuring and analyzing multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through one or more beams of light. The properties measured include the particle`s relative size and granularity or internal complexity and relative fluorescence intensity. After hydrodynamic focusing (produces a single stream of cells) cells are carried to the laser intercept. When these cells pass through the laser intercept, they scatter laser light. Light that is scattered in the forward direction is collected by a lens known as the forward scatter channel (FSC). The FSC intensity nearly equates to the particle`s size and can be used to distinguish between cellular debris and living cells. Light measured perpendicular to the excitation line is called side scatter. The side scatter channel (SSC) provides information about cell complexity or granularity.<br />
Fluorescent labeling allows investigation of cellular structure and functions. Flow cytometers use distinct fluorescence (FL-) channels to detect light emitted. The detection of fluorescent proteins in cells allows to monitor gene expression and to identify fluorescently labeled particles. <br />
There are a lot of fluorescent substances with potential applications in flow cytometry. The most frequently used molecule is the green fluorescent protein (GFP), a biological molecule derived from the jellyfish Aequorea victoria. Among GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive and uniquely quenched by chloride ions (Cl-). Found in the Registry of Standard Biological Parts, we used mVenus (BBa_I757008) as our desired gene of interest which contains a novel mutation at position F46L. SEYFP-F46L (Venus) folds well and forms the chromophore efficiently at 37°C (Nagai et al. 2002). The usage of fluorescent molecules as fusion proteins allows checking the transduction efficiency by determining the fluorescent intensity of YFP in transduced cells. GFP shows excitation and emission maxima at 489nm and 509nm, respectively. SEYFP-F46L`s peak excitation and emission wavelengths are 515nm and 528nm. Both GFP and SEYFP-F46L can be excited with a 488 nm blue laser and detected on FL 1.<br />
The number of fluorescent proteins that can be detected depends on the instruments and lasers available to the user. The Flow Cytometer CyAn ADP 9 Color from Beckman Coulter (Krefeld, Germany) is equipped with a 488 nm and a 405 nm laser and a 642nm diode which allows the detection of fluorescence of different fluorochromes. We used the 488 nm laser to excite mVenus (YFP) and the fluorescent channel 1 (FL 1) to detect light emitted. <br />
<br />
<br />
Figure 47: Laser Light Source to excite different Fluorochromes and the adapted fluorescent channels to detect light emitted (Beckmann Coulter 2008).<br />
<br />
<br />
<br />
Data analysis was carried out using Summit 4.3 (Beckman Coulter) software. Forward and side scatter light gating were used to exclude dead cells and debris (Fig. 5). A minimum of 10.000 events was collected for each gate and histogram, respectively.<br />
<br />
Analytical gates were set such that 1% or fewer of negative control cells fell within the positive region (Fig.6 left). The same gate was used to detect the YFP-Expression of transduced cells (Fig.6 right). For transduction we use human tumor cell lines (HT1080 and A431). YFP expression can be correlated with the transduction efficiency of the viral vectors by monitoring measured fluorescence.<br />
<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:13:16Z<p>Achim: /* A431 */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
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<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
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For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
=====Overview=====<br />
The A431 cells belong to the fibroblasts and the cell line was established from an epidermal carcinoma of a vulva. The main purpose of fibroblasts is to maintain the structure of connective tissues by continuously secreting precursors of the extracellular matrix. They are the most common cells in connective tissue in animals. The A431 cells show an epithelial morphology, have been used for a lot of different studies in cellbiology and are naturally devoid of a potent tumor suppressor and transcription factor: p53 protein (p53His273 mutation). <br />
The extreme expression of EGF receptors by this cell line is due, at least partly, to the amplification of EGF receptor DNA sequences (30-fold). Normal human cells exhibit a EGF receptor density ranging from 40.000 to 100.000 receptors/cell whereas the A431 cell line has 3x106 receptors/cell. (Carpenter & Cohen 1979) (Shimizu et al. 1984) (Panksepp et al. 1984)<br />
<br />
==Flow Cytometry==<br />
===Overview===<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:09:31Z<p>Achim: /* MTT Assay */</p>
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<h1>Methods</h1><br />
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<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<td width="489" valign="top"><br />
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<b>genotype</b><br />
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</p><br />
</td><br />
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<td width="130" valign="top"><br />
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BL21<br />
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</p><br />
</td><br />
<td width="489" valign="top"><br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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</p><br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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</p><br />
</td><br />
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<td width="130" valign="top"><br />
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XL10-Gold<br />
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</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
==Flow Cytometry==<br />
===Overview===<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
===Overview===<br />
===Protocol===<br />
<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:08:40Z<p>Achim: /* Flow Cytometry */</p>
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<h1>Methods</h1><br />
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=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
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Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
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===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
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==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
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The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
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After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
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===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
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==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
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===AFM===<br />
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Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
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===EM===<br />
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Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
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Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
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===Resources===<br />
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1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
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==ITR cloning==<br />
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[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
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As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
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<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
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==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
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===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
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=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
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The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
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PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
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Link to NEB: http://www.neb.com<br />
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===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
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As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
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Link to Agilent: http://www.genomics.agilent.com<br />
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===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
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===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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<b>genotype</b><br />
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BL21<br />
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<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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XL1-blue<br />
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<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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XL10-Gold<br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
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===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
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As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
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===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
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===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
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==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
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The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
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Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
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Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
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For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
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=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
==Flow Cytometry==<br />
===Overview===<br />
===Sample Preparation for Flow Cytometry===<br />
===Cell Staining for Flow Cytometry===<br />
<br />
==MTT Assay==<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:05:56Z<p>Achim: /* Flow Cytometry */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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</p><br />
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<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
<p><br />
</p><br />
</p><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
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<br />
</p><br />
</p><br />
</div><br />
</html><br />
===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
==Flow Cytometry==<br />
==MTT Assay==<br />
==Quantitative real-time PCR==<br />
===SYBR Green===<br />
===Protocols===<br />
====Genomic titer====<br />
====Infectious titer====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T22:03:43Z<p>Achim: /* A431 */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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<p><br />
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<table border="1" cellspacing="0" cellpadding="0"><br />
<tbody><br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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</p><br />
</p><br />
</td><br />
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<tr><br />
<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
<p><br />
</p><br />
</p><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
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<br />
</p><br />
</p><br />
</div><br />
</html><br />
===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
==Flow Cytometry==<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T21:58:31Z<p>Achim: /* Cell Lines */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
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<h1>Methods</h1><br />
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<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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<b>Cell strain</b><br />
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</td><br />
<td width="489" valign="top"><br />
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<b>genotype</b><br />
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</p><br />
</p><br />
</td><br />
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<td width="130" valign="top"><br />
<p><br />
BL21<br />
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</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
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</p><br />
</p><br />
</td><br />
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<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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</p><br />
</p><br />
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</td><br />
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</table><br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
====HT1080 Cells====<br />
====A431====<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T21:55:50Z<p>Achim: /* Passaging of AAV-293 Cells */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
<html><br />
<div><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
<p><br />
</p><br />
<table border="1" cellspacing="0" cellpadding="0"><br />
<tbody><br />
<tr><br />
<td width="619" colspan="2" valign="top"><br />
<p><br />
Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL10-Gold<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
<p><br />
</p><br />
</p><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p><br />
<p><br />
<br />
</p><br />
</p><br />
</div><br />
</html><br />
===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
<br><br><br />
<br />
===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
<br />
As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
<br />
===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
<br />
==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
<br />
The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
<br />
Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
<br />
Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
<br />
For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
<br />
=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
=====Transfecting the AAV-293 Cells=====<br />
Stratagene recommends a calcium phosphate-based protocol, usually resulting in the production of titers ≥ 107 particles/ml when AAV-293 cells were transfected.<br />
To achieve high titers, it is important that AAV-293 cells are healthy and plated at optimal densitiy. It should be taken care to avoid clumping of the cells during passaging and plating for transfection.<br />
Required materials and chemicals: 0.3 M CaCl2, 2x HBS-Buffer pH 7.1, autoclaved deionized (Millipore) water, 1.5 ml Eppi-tubes<br />
#Inspect the host cells that were split two days before; they should be approx. 70-80 % confluent<br />
#Remove the plasmids to be co-transfected from storage at -20 °C. Adjust the concentration of each plasmid to 1 µg/µl in sterile/autoclaved Millipore water.<br />
#Pipet the required volume of each of the plasmid DNA solution (5 µg of each plasmid) into an 1.5 ml tubes. Fill up to 300 µl with sterile Millipore water.<br />
#Add 300 µl of 0.5 M CaCl2 and mix gently.<br />
#Pipet 600 µl of 2x HBS into a 15 ml falcon.<br />
#Vortex the falcon gently while pipetting the DNA/CaCl2 solution dropwise into the falcon.<br />
#Incubate 20 minutes (precipitate formation).<br />
#Apply the DNA/CaCl2/2x HBS solution dropwise to the plate of cells (10 cm dish).<br />
#Return the cells in a 37 °C incubator at 5 % CO2 for 6 h.<br />
#After incubation remove the medium, wash once with PBS and replace it with 10 ml of fresh DMEM growth medium.<br />
#Return the cells back to the 37 °C incubator for an additional 66-72 h.<br />
<br />
=Protocols; Standard Operating Procedures=<br />
<br />
== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>Achimhttp://2010.igem.org/Team:Freiburg_Bioware/Project/MethodsTeam:Freiburg Bioware/Project/Methods2010-10-27T21:52:56Z<p>Achim: /* Establishing AAV-293 Cultures from Frozen Cells */</p>
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<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Methods</h1><br />
</html><br />
<br />
=Method Development=<br />
==Purification of AAV particles==<br />
===IMAC purification via Viral Brick: The Histidin Affinity Tag===<br />
<br />
<br />
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.<br />
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991)<br />
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.<br />
<br />
<br />
===Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide===<br />
The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). <br />
The purified BirA biotin ligase that was kindly provided by Avidity.<br />
<br />
==Experiences from under the hood: Cell Culture==<br />
===Optimizing the transfection protocol===<br />
[[Image:Freiburg10 cellculture1.jpg|Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers|thumb|right|250px]]<br />
The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined.<br />
One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. <br />
Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol.<br />
After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive. <br />
<br />
The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.<br />
<br />
After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.<br />
<br />
===Flow Cytometry Analysis===<br />
[[Image:Freiburg10 cellculture2.jpg|Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry|thumb|right|250px]]<br />
After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol.<br />
As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Imaging AAV: EM/AFM pictures==<br />
To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:<br />
<br />
===AFM===<br />
<gallery><br />
Image:Freiburg10 AAV afm.png|AFM picture: Phase <br />
Image:Freiburg10 AAV afm2.png|AFM picture: Height<br><br />
</gallery><br />
<br />
<br />
<br />
===EM===<br />
<br />
<gallery><br />
Image:Freiburg10_EM1.png|EM picture 1<br />
Image:Freiburg10 EM2.jpg|EM picture 2<br />
</gallery><br />
<br />
<br />
Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.<br />
<br />
===Resources===<br />
<br />
1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.<br />
<br />
==ITR cloning==<br />
<br />
[[Image:Freiburg10_diarylastgel.png|thumb|right|200px|Test Digestion of our final ITR BioBricks]]<br />
<br />
As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a <u>[https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary complex cloning strategy]</u> that finally led to functional ITR motives in the rfc10 standard.<br />
<br />
<br><br />
<br />
<u>See also: [https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary Hannas' ITR Diary]</u><br />
<br />
<br />
<br><br><br />
<br />
==Serum-free cell culture medium==<br />
===Introduction===<br />
<!--[[Image:Freiburg10_Medium.png|thumb|right|200px]]--><br />
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).<br><br />
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. <br><br />
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.<br><br />
<br />
===Testing serum free medium===<br />
Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.<br><br />
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.<br><br />
===Results===<br />
Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.<br />
<br />
<br />
=Established Methods=<br />
==Cloning==<br />
===Polymerase Chain Reaction===<br />
The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.<br />
<br />
The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew. <br />
<br />
PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).<br />
<br />
Link to NEB: http://www.neb.com<br />
<br />
===Site-Directed Mutagenesis===<br />
The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.<br />
<br />
As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.<br />
<br />
Link to Agilent: http://www.genomics.agilent.com<br />
<br />
===Ligation===<br />
[[Image:Freiburg10 Ligation.png|right|thumb|300px|scematic representation of the idempotent cloning principle]]<br />
Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP.<br />
This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
===Transformation===<br />
Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.<br />
<html><br />
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Table 2: Genotypic characterization of bacterial strains used for tramsformation.<br />
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</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
<b>Cell strain</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<b>genotype</b><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
BL21<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>E. coli </i><br />
B F<sup>–</sup><i>dcm ompT hsdS</i>(r<sub>B</sub><sup>–</sup> m<sub>B</sub><sup>–</sup>) <i>gal</i><br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="130" valign="top"><br />
<p><br />
XL1-blue<br />
<p><br />
</p><br />
</p><br />
</td><br />
<td width="489" valign="top"><br />
<p><br />
<i>recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac </i><br />
[F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10 </i>(Tet<sup>r</sup>)]<br />
<p><br />
</p><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
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Tetr<sup>r</sup><i>Δ</i>(<i>mcrA</i>)<i>183 </i><i>Δ</i>(<i>mcrCB-hsdSMR-mrr</i>)<i>173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac </i><br />
Hte [F´ <i>proAB lacI</i><sup>q</sup><i>Z</i><i>Δ</i><i>M15 </i>Tn<i>10</i> (Tet<sup>r</sup>) Amy Cam<sup>r</sup>]<br />
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===PCR purification===<br />
PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used.<br />
Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)<br />
===Hybridisation of Oligos===<br />
Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.<br />
===Fill-in-reactions===<br />
[[Image:Klenow.jpg|right|thumb|250px|Scematic depiction of a Fill-in reaction using Klenow fragment]]<br />
Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.<br />
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===DNA gel electrophoresis===<br />
[[Image:Freiburg10 Ladder.png|right|thumb|200px|GeneRuler DNA ladder mix provided by Fermentas]]<br />
Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com <br />
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As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).<br />
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===Vector Dephosphorylation===<br />
A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. <br />
[http://www.neb.com]. <br />
Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.<br />
===Gel Extraction===<br />
This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).<br />
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===Verification of Correct BioBrick Part Assembly===<br />
After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.<br />
===Test digestion===<br />
It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V. <br />
===Colony PCR===<br />
Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.<br />
===Purification of Plasmid DNA===<br />
====Miniprep====<br />
We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)<br />
====Midiprep====<br />
The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)<br />
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==Cell Culture==<br />
===Cell Lines===<br />
====HEK 293 Cells====<br />
=====Overview=====<br />
The 293 cell line was derived from primary cultures of human embryonic kidney (HEK) cells with sheared fragments of adenovirus (Ad) 5 DNA (Graham et al. 1977). HEK 293 cells contain the nucleotides 1-4344 of Ad5 which are located within the pregnancy-specific ß-1-glycoprotein 4 (PSG 4) gene. The transforming region of the human adenovirus contains the early region (E1), comprising two transcription units, E1a and E1b, whose products are essential and sufficient for mammalian cell transformation by adenoviruses (Louis et al. 1997). Because 293 cells express E1 gene products they are extensively used for the production of E1-deleted Ad viruses.<br />
Adeno-associated viruses (AAVs) belong to the family of Parvoviridae, being one of the smallest single-stranded and non-enveloped DNA viruses. AAVss are replication-deficient and have required co-infection with a helper adeno- or herpes virus for productive infection.<br />
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The AAV Helper-free system takes advantage of the identification of the specific adenovirus gene products that mediate AAV replication and allows the production of infectious recombinant human adeno-asscociated virus-2 (AAV-2) virions without the use of a helper virus (AAV Helper-free System Instruction Manual, Agilent Technologies).<br />
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Stratagene recommends preparing Adeno-associated virus stocks using the AAV-293 cell line.The AAV-293 cells are derived from the commonly used HEK293 cell line, but produce higher viral titers. These cells also allow production of infectious viral particles when cells are co-transfected with the three AAV Helper-Free System plasmids (ITR containing plasmid, pAAV_RC and pHelper) because the adenovirus E1 gene product is stably expressed in AAV-293 cells.<br />
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Thus the AAV-293 cell line is specifically selected for high levels of AAV production in a Helper-Free System and offers several advantages over common HEK293 cells.<br />
=====Establishing AAV-293 Cultures from Frozen Cells=====<br />
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For safe treatment of cells, the following steps have to be carried out under sterile conditions. Required materials and chemicals are: DMEM (Dulbecco`s Modified Eagle Medium 1x, Invitrogen, Darmstadt, Germany), PBS (Dulbecco`s PBS (1x) w/o Ca and Mg, PAA Laboratories GmbH, Pasching, Austria), T75 flask (Nunc, 75 cm2 nunclon treated flask, blue filter cap, Roskilde, Denmark), 15 ml falcon, pipett tips<br />
#Thaw frozen cells within 1-2 minutes by gentle agitation in a 37 °C water bath<br />
#Transfer the thawed cells suspension into the 15 ml falcon containing 10 ml of DMEM<br />
#Collect cells by centrifugation at 200 x g for 5 minutes at room temperature (Centrifuge 5702, Eppendorf, Hamburg, Germany)<br />
#Remove supernatant and resuspend the cells in 3 ml of fresh DMEM by gently pipetting up and down<br />
#Transfer the 3 ml of cell suspension to a T75 flask containing 17 ml of DMEM<br />
#Place the cells in a 37 °C incubator at 5 % CO2.<br />
#Monitor cell density daily. Cells should be passaged when the culture reaches 50 % confluence.<br />
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=====Passaging of AAV-293 Cells=====<br />
Required materials and chemicals: DMEM, PBS, Trypsin-EDTA 0,25 % (Invitrogen, Darmstadt, Germany), T75 flask, 15 ml falcon, pipett tips<br />
#Prewarm the DMEM to 37 °C in a water bath and trypsin-EDTA solution at room temperature<br />
#Remove the medium and wash cells once with 10ml of phosphate-buffer saline (PBS)<br />
#Trypsinize the cells for 1-1.5 minutes in 1 ml of trypsin-EDTA solution<br />
#Dilute the cells with 10 ml DMEM to inactivate the trypsin and detach the remaining cells by soft resuspending <br />
#Transfer cells into a 15 ml falcon<br />
#Collect cells by centrifugation at 200 g for 5 minutes<br />
#Calculate the cell amount per ml via Neubauer cell chamber<br />
##Mix 95 µl Tryptan Blue Stain 0,4 % (Lonza, Walkersville, USA) with 5 µl of the cell suspension<br />
##Mix gently by pipetting up and down<br />
##Pipet the solution into the Neubauer chamber <br />
##Tryptan stains dead cells blue, living cells appear as white<br />
##Counting of all living cells in four big squares<br />
##Calculate the amount of living cells per ml with the help of following formula: (counted cells/ 4 ) * 2.2 * 20 * 10.000<br />
#Calculate the cell amount per T75 flask (1.500.000 cells/20 ml DMEM) and transfer the cell suspension to a T75 flask containing fresh DMEM. Place the cells in a 37 °C incubator at 5 % CO2.<br />
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=Protocols; Standard Operating Procedures=<br />
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== Standard Protocol: Cloning ==<br />
[[Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf]]<br><br />
[[Media:Split_cellculture.pdf]]<br><br />
[[Media:Freeze_cellculture.pdf]]<br><br />
[[Media:production of competent E.coli.pdf]]<br><br />
[[Media:Freiburg10_Transfection_protocoll.pdf]]<br><br />
[[Media:Freiburg10 Thawing cells.pdf]]<br><br />
[[Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf]]<br><br />
[[Media:Freiburg10 Endotoxinfreie Midi.pdf]]<br><br />
[[Media:Freiburg10_LB+Agar.pdf]]<br><br />
[[Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf]]<br><br />
[[Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf]]<br><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Achim