Team:Freiburg Bioware/Project/Results/Modularization Vector Plasmid

From 2010.igem.org

(Difference between revisions)
(New page: {{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}} <html> <h2>Modularization GOI/Vector Plasmid</h2> <h3>Introduction to Modularization</h...)
 
(36 intermediate revisions not shown)
Line 1: Line 1:
-
{{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}}
+
{{:Team:Freiburg_Bioware/Head}}
 +
{{:Team:Freiburg_Bioware/jquery}}
 +
{{:Team:Freiburg_Bioware/menu_home}}
 +
 
<html>
<html>
-
<h2>Modularization GOI/Vector Plasmid</h2>
+
<h1>Modularization</h1>
-
<h3>Introduction to Modularization</h3>
+
<br>
-
<p class="MsoNormal">Producing recombinant virus particles
+
<h2>Contents</h2>
-
for therapeutical
+
<h3>Modularization of the vectorplasmid</h3>
-
means is, besides specifically target cells, purification and
+
<p class="MsoToc2"><a href="#_Toc275800680"><span lang="EN-US">Introduction
-
quantification
+
to
-
assays of AAV-2, one intention of the Virus Construction Kit provided
+
Modularization of Vectorplasmid</span><span
-
by the
+
style="color: windowtext; display: none; text-decoration: none;">.
-
iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the
+
</span><span
-
complex
+
style="color: windowtext; display: none; text-decoration: none;">1</span></a></p>
-
components of AAV-2 were extracted and redesigned to match the iGEM
+
<p class="MsoToc3"><a href="#_Toc275800681"><span lang="EN-US">Recombinant
-
standard.
+
-
Functional activity was tested in cell culture.</p>
+
-
<p class="MsoNormal">Differing from the wildtype AAV-2
+
-
genome, the Helper Free
+
-
System provided by Stratagene comprises three plasmids and a
+
-
specialized
+
-
production cell line. AAV-293 cells derived from the HEK cell line
+
-
express the
+
-
stably integrated E1A and E1B helper proteins for efficient virus
+
-
production. The
+
-
plasmid containing the inverted terminal repeats (ITRs) is encapsidated
+
-
into
+
-
the preformed capsids after production of single-stranded DNA therefore
+
-
also
+
-
known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i>
+
-
intron and the hGH
+
-
terminator signal are flanked by the ITRs and serve in the host cell
+
-
for
+
-
regulation of transgene expression. In addition to that, the plasmid
+
-
coding for
+
-
the Rep and Cap proteins (pRC) can be provided <i>in trans</i>
+
-
leading to a
+
-
layer of specificity due to the fact that the two genes are not
+
-
packaged into
+
-
the capsid since lacking of the ITRs impairs encapsidation. Another
+
-
advantage
+
-
of the Helper Free System can be attributed to cotransfection of
+
-
another helper
+
-
plasmid (pHelper), which provides the necessary proteins normally
+
-
obtained by
+
-
superinfection with helper viruses such as adenovirus or herpes simplex
+
-
virus.
+
-
These helper genes are required for full viral assembly by regulating
+
-
gene
+
-
expression of Rep and Cap proteins.</p>
+
-
<h3>Recombinant and Modular Vector Plasmid Carrying GOI</h3>
+
-
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010
+
-
provides a modular Virus
+
-
Construction Kit for therapeutical applications, quantification assays
+
and
and
-
purification approaches depending on capsid modifications and the gene
+
Modular Vectorplasmid Carrying GOI</span><span
-
of
+
style="color: windowtext; display: none; text-decoration: none;">
-
interest flanked by the inverted terminal repeats (ITRs. In order to
+
</span><span
-
produce
+
style="color: windowtext; display: none; text-decoration: none;">2</span></a></p>
-
BioBrick-compatible standardized biological parts, we reengineered the
+
<p class="MsoToc3"><a href="#_Toc275800682"><span lang="EN-US">Cloning
-
plasmids
+
-
and added new components for gene therapy approaches and analysis of
+
-
biological
+
-
activity of assembled BioBrick parts. Each element required for intact
+
and
and
-
functional plasmids comprising the ITRs, a promoter, a putative
+
Combination Strategies for the Vectorplasmid</span><span
-
enhancer
+
style="color: windowtext; display: none; text-decoration: none;">.
-
element and the hGH terminator was PCR amplified and fused together <i>de
+
</span><span
-
novo</i>.
+
style="color: windowtext; display: none; text-decoration: none;">3</span></a></p>
-
As shown in Figure 1, the vectorplasmid was assembled with the produced
+
<p class="MsoToc3"><a href="#_Toc275800683"><span lang="EN-US">Testing
-
BioBricks consisting of the left and right ITR (BBa_K404100 and
+
functionality of Assembled Vectorplasmid</span><span
-
BBa_K404101), a
+
style="color: windowtext; display: none; text-decoration: none;">.
-
promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin
+
</span><span
-
intron (BBa_K404107),
+
style="color: windowtext; display: none; text-decoration: none;">7</span></a></p>
-
the gene of interests (fluorescent proteins mVenus: BBa_I757008 and
+
<p class="MsoToc4"><a href="#_Toc275800684"><span lang="EN-US">Fluorescence
-
mCherry: BBa_J06504,
+
Microscopy of Target Cells Demonstrates GOI Expression</span><span
-
suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39: BBa_K404315 and CD:
+
style="color: windowtext; display: none; text-decoration: none;">.
-
BBa_K404112)
+
</span><span
-
and the hGH terminator (BBa_K404108).</p>
+
style="color: windowtext; display: none; text-decoration: none;">7</span></a></p>
 +
<p class="MsoToc4"><a href="#_Toc275800685"><span lang="EN-US">Analysis
 +
of Target
 +
Cells by Flow Cytometry demonstrates GOI Expression</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">8</span></a></p>
 +
<p class="MsoToc5"><a href="#_Toc275800686"><span lang="EN-US">Influence
 +
of hGH
 +
terminator BioBrick on GOI Expression</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">8</span></a></p>
 +
<p class="MsoToc5"><a href="#_Toc275800687"><span lang="EN-US">Influence
 +
of <i>Beta-globin</i>
 +
intron Biobrick on GOI Expression</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">11</span></a></p>
 +
<p class="MsoToc5"><a href="#_Toc275800688"><span lang="EN-US">Functionality
 +
of the
 +
Full Assembled Vectorplasmid Demonstrated by GOI Expression</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">14</span></a></p>
 +
<p class="MsoToc3"><a href="#_Toc275800689"><span lang="EN-US">Conclusion</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">16</span></a></p>
 +
<p class="MsoToc3"><a href="#_Toc275800690"><span lang="EN-US">References</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">17</span></a></p>
 +
<p class="MsoNormal"><a name="_Toc275797953"><b>&nbsp;</b></a></p>
 +
<h3>Modularization of the RepVP123 plasmids</h3>
 +
<div class="WordSection1">
 +
<p class="MsoToc2"><a href="#_Toc275885921"><span lang="EN-US">Overview
 +
of RepVP123
 +
plasmid</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">2</span></a></p>
 +
<p class="MsoToc2"><a href="#_Toc275885922"><span lang="EN-US">Modularization:
 +
Overview</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">..
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">2</span></a></p>
 +
<p class="MsoToc3"><a href="#_Toc275885923"><span lang="EN-US">Modularization:
 +
Removing iGEM restriction sites and establishing loop insertion
 +
capability</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">3</span></a></p>
 +
<p class="MsoToc4"><a href="#_Toc275885924"><span lang="EN-US">Modifications
 +
in Rep</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">3</span></a></p>
 +
<p class="MsoToc4"><a href="#_Toc275885925"><span lang="EN-US">Modifications
 +
in
 +
VP123</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">4</span></a></p>
 +
<p class="MsoToc3"><a href="#_Toc275885926"><span lang="EN-US">Modularization:
 +
Adapting pSB1C3 to loop insertions – pSB1C3_001</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">7</span></a></p>
 +
<p class="MsoToc4"><a href="#_Toc275885927"><span lang="EN-US">Turning-off
 +
natural
 +
tropism: HSPG-knock-out</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">9</span></a></p>
 +
<p class="MsoToc2" style="margin-left: 0cm; text-indent: 0cm;">&nbsp;</p>
 +
<span
 +
style="font-size: 11pt; line-height: 115%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US"><br style="page-break-before: always;" clear="all">
 +
</span>
 +
<h2><a name="_Toc275800680">Introduction to
 +
Modularization of vector
 +
plasmid</a></h2>
 +
<br>
 +
<p class="MsoNormal"><span lang="EN-US">Producing
 +
recombinant virus
 +
particles for
 +
therapeutical means is, besides specifically target cells, purification
 +
and
 +
quantification assays of AAV-2, one intention of the Virus Construction
 +
Kit
 +
provided by the iGEM team Freiburg_Bioware 2010. For obtaining a
 +
modular
 +
toolkit, the complex components of AAV-2 were extracted and redesigned
 +
to match
 +
the iGEM standard. Functional activity was tested in cell culture.</span></p>
 +
<p class="MsoNormal"><span lang="EN-US">Differing
 +
from the wildtype
 +
AAV-2 genome,
 +
the Helper Free System provided by Stratagene comprises three plasmids
 +
and a
 +
specialized production cell line. AAV-293 cells derived from the HEK
 +
cell line
 +
express the stably integrated E1A and E1B helper proteins for efficient
 +
virus
 +
production. The plasmid containing the inverted terminal repeats (ITRs)
 +
is
 +
encapsidated into the preformed capsids after production of
 +
single-stranded DNA
 +
therefore also known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i>
 +
intron and the hGH terminator signal are flanked by the ITRs and serve
 +
in the
 +
host cell for regulation of transgene expression. In addition to that,
 +
the
 +
plasmid coding for the Rep and Cap proteins (pRC) can be provided <i>in
 +
trans</i>
 +
leading to a layer of specificity due to the fact that the two genes
 +
are not
 +
packaged into the capsid since lacking of the ITRs impairs
 +
encapsidation. Another
 +
advantage of the Helper Free System can be attributed to cotransfection
 +
of
 +
another helper plasmid (pHelper), which provides the necessary proteins
 +
normally obtained by superinfection with helper viruses such as
 +
adenovirus or herpes
 +
simplex virus. These helper genes are required for full viral assembly
 +
by
 +
regulating gene expression of Rep and Cap proteins.</span></p>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800681"></a><a
 +
name="_Toc275797954"><span lang="EN-US"><span
 +
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
 +
lang="EN-US">Recombinant and Modular Vectorplasmid Carrying
 +
GOI</span></a></h3>
 +
<p class="MsoNormal"><span lang="EN-US">The
 +
iGEM team Freiburg_Bioware
 +
2010 provides
 +
a modular Virus Construction Kit for therapeutical applications,
 +
quantification
 +
assays and purification approaches depending on capsid modifications
 +
and the
 +
gene of interest flanked by the inverted terminal repeats (ITRs. In
 +
order to
 +
produce BioBrick-compatible standardized biological parts, we
 +
reengineered the
 +
plasmids and added new components for gene therapy approaches and
 +
analysis of
 +
biological activity of assembled BioBrick parts. Each element required
 +
for
 +
intact and functional plasmids comprising the ITRs, a promoter, a
 +
putative
 +
enhancer element and the hGH terminator was PCR amplified and fused
 +
together <i>de
 +
novo</i>.</span><span lang="EN-US"> As shown
 +
in </span><span lang="EN-US">Figure 1</span><span lang="EN-US">, the
 +
vectorplasmid was
 +
assembled
 +
with the produced BioBricks consisting of the left and right ITR
 +
(<a href = "http://partsregistry.org/Part:BBa_K404100" target="blank" > BBa_K404100</a>
 +
and <a href = "http://partsregistry.org/Part:BBa_K404101" target="blank" > BBa_K40410</a>), a promoter (pCMV :<a href = "http://partsregistry.org/Part:BBa_K404102" target="blank" > BBa_K404102</a> or phTERT:
 +
<a href = "http://partsregistry.org/Part:BBa_K404106" target="blank" > BBa_K404106</a>)) , the
 +
beta-globin intron (<a href = "http://partsregistry.org/Part:BBa_K404107" target="blank" > BBa_K404107</a>), the gene of interests (fluorescent
 +
proteins
 +
mVenus: <a href = "http://partsregistry.org/Part:BBa_I757008" target="blank" > BBa_I757008</a> and mCherry: <a href = "http://partsregistry.org/Part:BBa_J06504" target="blank" > BBa_J06504</a>, suicide genes mGMK_TK30:
 +
(<a href = "http://partsregistry.org/Part:BBa_K404112" target="blank" > BBa_K404112</a>,
 +
mGMK_SR39: <a href = "http://partsregistry.org/Part:BBa_K4043150" target="blank" > BBa_K404315</a> and CD: <a href = "http://partsregistry.org/Part:BBa_K404112" target="blank" > BBa_K404112</a>) and the hGH terminator
 +
(<a href = "http://partsregistry.org/Part:BBa_K404108" target="blank" > BBa_K404108</a>).</span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr>
+
<tr style="height: 522.25pt;">
-
      <td
+
<td style="padding: 0cm 5.4pt; width: 441.75pt; height: 522.25pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 81.95pt;"
+
valign="top" width="589">
-
width="109">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
id="Grafik 5"
-
align="center"><img id="Grafik 1"
+
src="http://2010.igem.org/wiki/images/a/ae/Freiburg10_Overview_BBa_Vectorplasmid.png"
-
src="Freiburg10_Modularization_GOI_02_files/image001.gif"
+
height="692" width="580"></p>
-
alt="Description: http://partsregistry.org/wiki/images/b/ba/Freiburg10_VectorplasmidBrick_1.png"
+
<p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure
-
height="83" width="102"></p>
+
</span><span lang="EN-US">1</span><span lang="EN-US">: </span><span
-
      </td>
+
style="font-weight: normal;" lang="EN-US">Overview of
-
      <td
+
the theoretical
-
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"
+
-
width="103">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Grafik 51"
+
-
src="Freiburg10_Modularization_GOI_02_files/image002.gif"
+
-
height="69" width="86"></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"
+
-
width="122">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><img id="Grafik 6"
+
-
src="Freiburg10_Modularization_GOI_02_files/image003.gif"
+
-
alt="Description: http://partsregistry.org/wiki/images/1/1e/Freiburg10_VectorplasmidBricks_4.png"
+
-
height="78" width="116"></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"
+
-
width="98">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><img id="Grafik 26"
+
-
src="Freiburg10_Modularization_GOI_02_files/image004.gif"
+
-
height="66" width="90"></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"
+
-
width="104">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+
-
align="left"><img id="Grafik 8"
+
-
src="Freiburg10_Modularization_GOI_02_files/image005.gif"
+
-
alt="Description: http://partsregistry.org/wiki/images/0/06/Freiburg10_VectorplasmidBricks_5.png"
+
-
height="80" width="96"></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"
+
-
width="105">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><img id="Grafik 4"
+
-
src="Freiburg10_Modularization_GOI_02_files/image006.gif"
+
-
alt="Description: http://partsregistry.org/wiki/images/1/18/Freiburg10_VectorplasmidBricks_2.png"
+
-
height="79" width="98"></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+
-
width="109">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 43"
+
-
src="Freiburg10_Modularization_GOI_02_files/image007.jpg"
+
-
height="80" width="79"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">Left ITR </span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">(</span><span
+
-
style="font-size: 9pt; color: black;" lang="DE">BBa_K404100</span><span
+
-
style="font-size: 9pt;" lang="DE">)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"
+
-
width="103">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 70"
+
-
src="Freiburg10_Modularization_GOI_02_files/image008.jpg"
+
-
height="80" width="80"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">pCMV</span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">(BBa_K404102)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"
+
-
width="122">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 61"
+
-
src="Freiburg10_Modularization_GOI_02_files/image009.jpg"
+
-
height="79" width="78"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><i><span style="font-size: 9pt;"
+
-
lang="DE">Beta-globin</span></i><span
+
-
style="font-size: 9pt;" lang="DE"> intron </span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">(</span><span
+
-
style="font-size: 9pt; color: black;" lang="DE">BBa_K404107</span><span
+
-
style="font-size: 9pt;" lang="DE">)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"
+
-
width="98">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 58"
+
-
src="Freiburg10_Modularization_GOI_02_files/image010.jpg"
+
-
height="79" width="79"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">mVenus (BBa_I757008)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"
+
-
width="104">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 64"
+
-
src="Freiburg10_Modularization_GOI_02_files/image011.jpg"
+
-
height="80" width="80"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">hGH terminator</span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">(BBa_K404108)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"
+
-
width="105">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 46"
+
-
src="Freiburg10_Modularization_GOI_02_files/image012.jpg"
+
-
height="79" width="79"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">Right ITR</span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">(BBa_K404101)</span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+
-
width="109">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"
+
-
width="103">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 67"
+
-
src="Freiburg10_Modularization_GOI_02_files/image013.jpg"
+
-
height="80" width="79"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">phTERT (BBa_K404106)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"
+
-
width="122">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"
+
-
width="98">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 109"
+
-
src="Freiburg10_Modularization_GOI_02_files/image014.jpg"
+
-
height="79" width="79"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">mCherry</span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">(BBa_J06504)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"
+
-
width="104">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"
+
-
width="105">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+
-
width="109">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"
+
-
width="103">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"
+
-
width="122">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"
+
-
width="98">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 10"
+
-
src="Freiburg10_Modularization_GOI_02_files/image015.jpg"
+
-
height="81" width="80"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">Cytosine deaminase (BBa_K404112)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"
+
-
width="104">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"
+
-
width="105">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+
-
width="109">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"
+
-
width="103">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"
+
-
width="122">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"
+
-
width="98">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 13"
+
-
src="Freiburg10_Modularization_GOI_02_files/image016.jpg"
+
-
height="79" width="80"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">mGMK_SR39</span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">(BBa_K404315)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"
+
-
width="104">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"
+
-
width="105">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+
-
width="109">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"
+
-
width="103">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"
+
-
width="122">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"
+
-
width="98">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Picture 7"
+
-
src="Freiburg10_Modularization_GOI_02_files/image017.jpg"
+
-
height="81" width="81"></span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">mGMK_TK30</span></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">(BBa_K404113)</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"
+
-
width="104">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"
+
-
width="105">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+
-
align="center"><span style="font-size: 9pt;"
+
-
lang="DE">&nbsp;</span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td colspan="6"
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
-
width="641">
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+
-
align="center"><span style="font-size: 9pt;"><img
+
-
id="Grafik 2057"
+
-
src="Freiburg10_Modularization_GOI_02_files/image018.gif"
+
-
height="116" width="555"></span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td colspan="6"
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
-
width="641">
+
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
-
name="_Ref275644946">Figure </a>1: <span
+
-
style="font-weight: normal;">Overview of the theoretical
+
sequence of each BioBrick provided within the Virus Construction Kit
sequence of each BioBrick provided within the Virus Construction Kit
for an intact and fully functional rAAV genome. The plasmid in the
for an intact and fully functional rAAV genome. The plasmid in the
Line 523: Line 224:
these constructs can be found under ‘Arming: Killing the tumor’ and
these constructs can be found under ‘Arming: Killing the tumor’ and
‘N-terminal fusion for Targeting’.</span></p>
‘N-terminal fusion for Targeting’.</span></p>
-
      </td>
+
</td>
-
    </tr>
+
</tr>
-
  </tbody>
+
</tbody>
</table>
</table>
-
<h3>Cloning and Combination Strategies for the Vectorplasmid </h3>
+
</div>
-
<p class="MsoNormal">Organization of the recombinant viral
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
DNA was modified
+
<h3><a name="_Toc275800682"></a><a name="_Toc275797955"><span
-
ensuring several layers of specificity to our systems including a
+
lang="EN-US">Cloning
 +
and Combination Strategies for the Vectorplasmid</span></a><span
 +
lang="EN-US"> </span></h3>
 +
<p class="MsoNormal"><span lang="EN-US">Organization
 +
of the recombinant
 +
viral DNA was
 +
modified ensuring several layers of specificity to our systems
 +
including a
tumor-specific promoter and suicide genes encoding prodrug convertases.
tumor-specific promoter and suicide genes encoding prodrug convertases.
In
In
-
order to modularize the rAAV sequence, each plasmid element (Figure 1)
+
order to modularize the rAAV sequence, each plasmid element (</span><span
-
was PCR-amplified and cloned into the iGEM standard plasmid pSB1C3.
+
lang="EN-US">Figure 1</span><span lang="EN-US">)
-
Furthermore, the iGEM
+
was PCR-amplified and
-
team Freiburg_Bioware 2010 performed three site-directed mutagenesis in
+
cloned into
 +
the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team
 +
Freiburg_Bioware
 +
2010 performed three site-directed mutagenesis in the gene of interest
 +
TK30 (<a href = "http://partsregistry.org/Part:BBa_K404109" target="blank" > BBa_K404109</a>)
 +
and cytosine deaminase (</span><span
 +
style="font-size: 9pt; line-height: 200%;" lang="EN-US"><a href = "http://partsregistry.org/Part:BBa_K404112" target="blank" > BBa_K404112</a></span><span
 +
lang="EN-US">) for deletion of PstI and
 +
NgoMIV iGEM site (for further information see the results page of
 +
‘Arming –
 +
Killing the tumor’). Since the inverted terminal repeats (ITRs) are
 +
GC-rich
 +
regions forming T-shaped hairpins during replication, PCR amplification
 +
was not
 +
possible. Hence a cloning strategy was developed by the iGEM team
 +
Freiburg in
 +
order to provide BioBrick-compatible ITRs (see ‘Method Development of
 +
Cloning
 +
Strategy for ITRs’).</span></p>
 +
<p class="MsoNormal"><span lang="EN-US">In </span><span lang="EN-US">Figure
 +
2</span><span lang="EN-US"> the schematic overview
 +
of the
 +
modularization process can be seen which has been followed to conduct
the
the
-
gene of interest TK30 (BBa_K404109) and cytosine deaminase (<span
+
assembly steps required for functional vectorplasmids.</span></p>
-
style="font-size: 9pt; line-height: 200%;">BBa_K404112</span>)
+
-
for deletion of
+
-
PstI and NgoMIV iGEM site (for further information see the results page
+
-
of
+
-
‘Arming – Killing the tumor’). Since the inverted terminal repeats
+
-
(ITRs) are
+
-
GC-rich regions forming T-shaped hairpins during replication, PCR
+
-
amplification
+
-
was not possible. Hence a cloning strategy was developed by the iGEM
+
-
team
+
-
Freiburg in order to provide BioBrick-compatible ITRs (see ).</p>
+
-
<p class="MsoNormal">In Figure 2 the schematic overview of
+
-
the modularization
+
-
process can be seen which has been followed to conduct the assembly
+
-
steps
+
-
required for functional vectorplasmids.</p>
+
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr style="height: 26.15pt;">
+
<tr style="height: 26.15pt;">
-
      <td
+
<td style="padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"
+
valign="top" width="454">
-
valign="top" width="454">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+
id="Grafik 2"
-
id="Grafik 2"
+
src="http://2010.igem.org/wiki/images/3/39/Freiburg10_Overview_Assembly_GOI.png"
-
src="Freiburg10_Modularization_GOI_02_files/image019.gif"
+
alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
-
alt="Description: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
+
height="272" width="439"></p>
-
height="272" width="439"></p>
+
<p class="MsoCaption" style="text-indent: 0cm;"><a
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
name="_Ref275783119"><span lang="EN-US">Figure </span></a><span
-
name="_Ref275783119">Figure </a>2: <span
+
lang="EN-US">3</span><span lang="EN-US">: </span><span
-
style="font-weight: normal;">Assembly procedure for fusion
+
style="font-weight: normal;" lang="EN-US">Assembly procedure for
-
of BioBricks and composite parts to a fully assembled and functional
+
fusion of BioBricks and composite parts to a fully assembled and
-
plasmid coding for your gene of interest. This plasmid can be
+
functional plasmid coding for your gene of interest. This plasmid can
-
cotransfected with two helper plasmids providing protein for assembly
+
be cotransfected with two helper plasmids providing protein for
-
and encapsidating of the rAAV genome (your gene of interest) into the
+
assembly and encapsidating of the rAAV genome (your gene of interest)
-
capsids.</span></p>
+
into the capsids.</span></p>
-
      </td>
+
</td>
-
    </tr>
+
</tr>
-
  </tbody>
+
</tbody>
</table>
</table>
</div>
</div>
-
<p class="MsoNormal">&nbsp;</p>
+
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal">The iGEM team Freiburg_Bioware
+
<p class="MsoNormal"><span lang="EN-US">The
-
provides two examples
+
iGEM team Freiburg_Bioware
-
demonstrating the assembly procedure for constructing vectorplasmids.
+
provides two
-
The first
+
examples demonstrating the assembly procedure for constructing
-
representative example is the fusion of the BioBrick part <i>beta-globin</i>
+
vectorplasmids.
-
to
+
The first representative example is the fusion of the BioBrick part <i>beta-globin</i>
-
the composite parts containing the 5´ elements of the plasmids, which
+
to the composite parts containing the 5´ elements of the plasmids,
-
are left
+
which are
-
ITR and CMV or phTERT promoter, respectively.</p>
+
left ITR and CMV or phTERT promoter, respectively.</span></p>
-
<p class="MsoNormal" style="text-indent: 0cm;">As
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">As
-
shown in Figure 3 the theoretical
+
shown in </span><span lang="EN-US">Figure 3</span><span lang="EN-US">
-
cloning performed for assembling the BioBricks <i>beta-globin </i>intron
+
the theoretical cloning performed
 +
for assembling the BioBricks <i>beta-globin </i>intron
and
and
-
leftITR_CMV together can be observed. </p>
+
leftITR_CMV together
 +
can be observed. </span></p>
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr style="height: 23.95pt;">
+
<tr style="height: 23.95pt;">
-
      <td
+
<td style="padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"
+
valign="top" width="467">
-
valign="top" width="467">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+
id="Grafik 74"
-
id="Grafik 74"
+
src="http://2010.igem.org/wiki/images/e/e3/Freiburg10_TheoreticalCloning_ITR_CMV_beta.png"
-
src="Freiburg10_Modularization_GOI_02_files/image020.gif"
+
height="530" width="452"></p>
-
height="530" width="452"></p>
+
<p class="MsoCaption" style="text-indent: 0cm;"><a
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
name="_Ref275783160"><span lang="EN-US">Figure </span></a><span
-
name="_Ref275783160">Figure </a>3: <span
+
lang="EN-US">4</span><span lang="EN-US">:</span><span lang="EN-US"> </span><span
-
style="font-weight: normal;">Theoretical cloning of the
+
style="font-weight: normal;" lang="EN-US">Theoretical
 +
cloning of the
composite part leftITR_CMV to the <i>beta-globin</i>
composite part leftITR_CMV to the <i>beta-globin</i>
-
intron BioBrick leading to the plasmid leftITR_CMV_<i>beta-globin</i>
+
intron BioBrick
 +
leading to the plasmid leftITR_CMV_<i>beta-globin</i>
intron.</span></p>
intron.</span></p>
-
      </td>
+
</td>
-
    </tr>
+
</tr>
-
  </tbody>
+
</tbody>
</table>
</table>
</div>
</div>
-
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal">The plasmids were digested with both
+
<p class="MsoNormal"><span lang="EN-US">The
-
XbaI and PstI
+
plasmids were digested with
-
(beta-globin intron: <span
+
both XbaI
-
style="font-size: 9pt; line-height: 200%; color: black;">BBa_K404107</span>)
+
and PstI (beta-globin intron: </span><span
-
or SpeI and PstI (leftITR_CMV) and loaded on an agarose gel. As
+
style="font-size: 9pt; line-height: 200%; color: black;" lang="EN-US"><a href = "http://partsregistry.org/Part:BBa_K404107" target="blank" > BBa_K404107</a></span><span
-
demonstrated in
+
lang="EN-US">)
-
the preparative gel in Figure 4, the expected bands could be detected
+
or SpeI and
-
under UV
+
PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the
-
light and the extracted DNA could be successfully ligated. Each
+
preparative gel in </span><span lang="EN-US">Figure
-
assembly step
+
4</span><span lang="EN-US">,
-
for producing BioBrick intermediates was conducted following the same
+
the expected bands could be detected under UV light and the extracted
-
strategy.</p>
+
DNA could
 +
be successfully ligated. Each assembly step for producing BioBrick
 +
intermediates was conducted following the same strategy.</span></p>
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr style="height: 129.95pt;">
+
<tr style="height: 129.95pt;">
-
      <td
+
<td style="padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"
+
valign="top" width="417">
-
valign="top" width="417">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p>
+
lang="EN-US">&nbsp;</span></p>
-
      <p class="MsoNormal"
+
<p class="MsoNormal"
-
style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+
style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-
align="right"><img id="Grafik 77"
+
align="right"><img id="Grafik 77"
-
src="Freiburg10_Modularization_GOI_02_files/image021.gif"
+
src="http://2010.igem.org/wiki/images/4/49/Freiburg10_Cloning_Intermediate_GOI.png"
-
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"
+
alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"
-
height="194" width="397"></p>
+
height="194" width="397"></p>
-
      <p class="MsoCaption"
+
<p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure
-
style="text-align: left; text-indent: 0cm;" align="left">Figure
+
</span><span lang="EN-US">5</span><span lang="EN-US">: Assembly
-
4: Assembly intermediate in fusion of the vectorplasmids containing
+
intermediate in fusion of the vectorplasmids containing different
-
different promoters. <span style="font-weight: normal;">Fusion
+
promoters. </span><span style="font-weight: normal;" lang="EN-US">Fusion
of the BioBrick part <i>beta-globin</i> (</span><span
of the BioBrick part <i>beta-globin</i> (</span><span
-
style="color: black;">BBa_K404107</span><span
+
style="color: black;" lang="EN-US"><a href = "http://partsregistry.org/Part:BBa_K404107" target="blank" > BBa_K404107</a></span><span
-
style="font-weight: normal;">) intron to the composite parts
+
style="font-weight: normal;" lang="EN-US">) intron to
-
leftITR_pCMV and leftITR_phTERT, respectively, was performed following
+
the composite
-
the BioBrick assembly strategy by digesting the insert with PstI and
+
parts leftITR_pCMV and leftITR_phTERT, respectively, was performed
-
XbaI and the vectors with SpeI and PstI. The left lane shows the
+
following the BioBrick assembly strategy by digesting the insert with
-
expected fragment at around 560 bp which corresponds to the <i>beta-globin</i>
+
PstI and XbaI and the vectors with SpeI and PstI. The left lane shows
 +
the expected fragment at around 560 bp which corresponds to the <i>beta-globin</i>
intron fragment, in contrast to the two lanes in the center and on the
intron fragment, in contrast to the two lanes in the center and on the
right which correspond to linearized plasmids after digesting with
right which correspond to linearized plasmids after digesting with
Line 669: Line 390:
Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV,
Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV,
pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p>
pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p>
-
      </td>
+
</td>
-
    </tr>
+
</tr>
-
  </tbody>
+
</tbody>
</table>
</table>
</div>
</div>
-
<p class="MsoNormal">&nbsp;</p>
+
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal">Separated fragments were extracted
+
<p class="MsoNormal"><span lang="EN-US">Separated
-
using the Gel Extraction
+
fragments were
-
Kit provided by Qiagen (Hilden, Germany) and ligated with T4-ligase.
+
extracted using
-
After
+
the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated
-
ligation has been carried out, <i>E. coli</i> XL-1B cells
+
with
-
were transformed and
+
T4-ligase. After ligation has been carried out, <i>E. coli</i>
-
incubated over night at 37°C. Picking clones from the transformation
+
XL-1B
-
plate was
+
cells were
-
performed the following day and DYT medium was inoculated incubating
+
transformed and incubated over night at 37°C. Picking clones from the
-
overnight.
+
transformation plate was performed the following day and DYT medium was
-
Plasmid DNA was isolated and test digestion revealed that cloning was
+
inoculated incubating overnight. Plasmid DNA was isolated and test
-
successful obtaining the composite part leftITR_CMV_<i>beta-globin</i>
+
digestion
-
intron (BBa_K404117).</p>
+
revealed that cloning was successful obtaining the composite part
-
<p class="MsoNormal">Plasmid production incorporating all
+
leftITR_CMV_<i>beta-globin</i>
-
required elements for
+
intron (<a href = "http://partsregistry.org/Part:BBa_K404117" target="blank" > BBa_K404117</a>).</span></p>
-
transgene expression and genome encapsidation into empty viral capsids
+
<p class="MsoNormal"><span lang="EN-US">Plasmid
-
was
+
production
-
performed by fusing the downstream elements consisting of the hGH
+
incorporating all
-
terminator and
+
required elements for transgene expression and genome encapsidation
-
right ITR to the intermediate part providing the gene of interest and
+
into empty
-
the
+
viral capsids was performed by fusing the downstream elements
-
promoter fused to the left ITR. Figure 5 demonstrates the assembly
+
consisting of the
-
performed
+
hGH terminator and right ITR to the intermediate part providing the
 +
gene of
 +
interest and the promoter fused to the left ITR. </span><span
 +
lang="EN-US">Figure 5</span><span lang="EN-US">
 +
demonstrates the
 +
assembly performed
with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus
with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus
and
and
-
pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after
+
pSB1C3_hGH_rightITR (<a href = "http://partsregistry.org/Part:BBa_K404116" target="blank" > BBa_K404116</a>). The fragment obtained after
digestion on the
digestion on the
left lane fits to the hGH-terminator_rightITR length. The isolated
left lane fits to the hGH-terminator_rightITR length. The isolated
Line 708: Line 434:
the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i>
the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i>
intron_mVenus_hGH_rightITR
intron_mVenus_hGH_rightITR
-
(<span style="line-height: 200%; color: black;">BBa_K404124</span>).
+
(</span><span style="line-height: 200%; color: black;" lang="EN-US"><a href = "http://partsregistry.org/Part:BBa_K404124" target="blank" > BBa_K404124</a></span><span
-
</p>
+
lang="EN-US">).
 +
</span></p>
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr style="height: 136.9pt;">
+
<tr style="height: 136.9pt;">
-
      <td
+
<td style="padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"
+
valign="top" width="411">
-
valign="top" width="411">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-
style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+
align="right"><img id="Grafik 80"
-
align="right"><img id="Grafik 80"
+
src="http://2010.igem.org/wiki/images/9/93/Freiburg10_Cloning_Full_GOI.png"
-
src="Freiburg10_Modularization_GOI_02_files/image022.gif"
+
alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"
-
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"
+
height="184" width="397"></p>
-
height="184" width="397"></p>
+
<p class="MsoCaption" style="text-indent: 0cm;"><a
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
name="_Ref275784510"><span lang="EN-US">Figure </span></a><span
-
name="_Ref275784510">Figure </a>5<span
+
lang="EN-US">6</span><span style="font-weight: normal;" lang="EN-US">:
-
style="font-weight: normal;">: </span>Modularization
+
</span><span lang="EN-US">Modularization of
-
of the assembled vectorplasmid containing the phTERT promoter and
+
the assembled
-
mVenus as gene of interest.<span style="font-weight: normal;">
+
vectorplasmid containing the phTERT promoter and mVenus as gene of
-
Fusion of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus
+
interest.</span><span style="font-weight: normal;" lang="EN-US"> Fusion
-
part&nbsp; to the composite parts pSB1C3_hGH_rightITR was performed
+
of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part
-
following the BioBrick assembly strategy by digesting the insert with
+
to the composite parts pSB1C3_hGH_rightITR was performed following the
-
XbaI and PstI and the vector with SpeI and PstI. The left lane
+
BioBrick assembly strategy by digesting the insert with XbaI and PstI
-
corresponds to linearized plasmid after digesting with above mentioned
+
and the vector with SpeI and PstI. The left lane corresponds to
-
iGEM restriction sites whereas the right lane reveals an intensive band
+
linearized plasmid after digesting with above mentioned iGEM
-
at around 650 bp confirming the expected size of 657 bp of hGH_rITR. M,
+
restriction sites whereas the right lane reveals an intensive band at
 +
around 650 bp confirming the expected size of 657 bp of hGH_rITR. M,
GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin
GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin
intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p>
intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p>
-
      </td>
+
</td>
-
    </tr>
+
</tr>
-
  </tbody>
+
</tbody>
</table>
</table>
</div>
</div>
-
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal">Since cloning does not confirm
+
<p class="MsoNormal"><span lang="EN-US">Since
-
biological activity, we
+
cloning does not confirm
-
analyzed the plasmids and their functional components, hGH terminator
+
biological
-
and <i>beta-globin</i>
+
activity, we analyzed the plasmids and their functional components, hGH
-
intron, in cell culture. Assembled plasmids have been cotransfected,
+
terminator and <i>beta-globin</i> intron, in cell culture.
-
using
+
Assembled
-
AAV-293 cells, which provide the stable integrated E1A and E1B genes,
+
plasmids
-
with
+
have been cotransfected, using AAV-293 cells, which provide the stable
-
helper plasmids required for capsid assembly&nbsp; and genome
+
integrated E1A and E1B genes, with helper plasmids required for capsid
-
encapsidation (pRC and
+
assembly and genome encapsidation (pRC and pHelper) in a molar ratio of
-
pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus particles
+
1:1:1
-
containing the single stranded DNA were harvested 72-hours post
+
(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA
-
transfection
+
were
-
and HT1080 cells transduced with constant volumes of viral vectors.
+
harvested 72 hours post transfection and HT1080 cells transduced with
-
48-hours
+
constant
-
post infection; transduced cells expressing the gene of interest were
+
volumes of viral vectors. 48 hours post infection; transduced cells
-
analyzed
+
expressing
-
by flow cytometry. Facilitating and demonstrating the analysis of
+
the gene of interest were analyzed by flow cytometry. Facilitating and
-
functionality
+
demonstrating the analysis of functionality of the assembled plasmid,
-
of the assembled plasmid, mVenus was used in first place since
+
mVenus
-
fluorescent
+
was used in first place since fluorescent proteins enable facile
-
proteins enable facile visualization using fluorescent microscopy and
+
visualization
-
flow
+
using fluorescent microscopy and flow cytometry analysis.</span></p>
-
cytometry analysis.</p>
+
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800683"></a><a
-
<h3>Testing functionality of Assembled Vectorplasmid</h3>
+
name="_Toc275797956"><span lang="EN-US"><span
-
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span
+
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
-
lang="DE">Fluorescence</span>
+
lang="EN-US">Testing functionality of Assembled Vectorplasmid</span></a></h3>
-
Microscopy of Target Cells Demonstrates GOI Expression</h4>
+
<h4><a name="_Toc275800684"></a><a name="_Toc275797957"><span
-
<p class="MsoNormal">Qualitative analysis of mVenus
+
lang="EN-US">Fluorescence
-
expression by fluorescence
+
Microscopy of Target Cells Demonstrates GOI Expression</span></a></h4>
-
microscopy was conducted using Axio Observer Z1 showing that transduced
+
<p class="MsoNormal"><span lang="EN-US">Qualitative
-
HT1080
+
analysis of mVenus
-
cells and non-transduced cells could be easily distinguished. In Figure
+
expression
-
6 cells were excited with 505nm and fluorescence emission at 536nm was
+
by fluorescence microscopy was conducted using Axio Observer Z1 showing
-
detected. Therefore, successful infection of tumor cells by recombinant
+
that
-
viral
+
transduced HT1080 cells and non-transduced cells could be easily
-
particles carrying the assembled vectorplasmid coding for mVenus could
+
distinguished.
-
be
+
In </span><span lang="EN-US">Figure 6</span><span lang="EN-US"> cells
-
demonstrated. </p>
+
were excited
 +
with 505nm and fluorescence emission at 536nm was detected. Therefore,
 +
successful
 +
infection of tumor cells by recombinant viral particles carrying the
 +
assembled vectorplasmid
 +
coding for mVenus could be demonstrated. </span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr>
+
<tr>
-
      <td
+
<td style="padding: 0cm 5.4pt; width: 250.7pt;" valign="top"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 250.7pt;"
+
width="334">
-
valign="top" width="334">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">A</p>
+
lang="EN-US">A</span></p>
-
      <p class="MsoNormal"
+
<p class="MsoNormal"
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
align="center"><img id="Grafik 18"
+
align="center"><img style="width: 264px; height: 218px;" id="Grafik 18"
-
src="Freiburg10_Modularization_GOI_02_files/image023.jpg"
+
src="http://2010.igem.org/wiki/images/a/af/Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png"
-
alt="Description: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"
+
alt=""></p>
-
height="218" width="264"></p>
+
</td>
-
      </td>
+
<td style="padding: 0cm 5.4pt; width: 230.4pt;" valign="top"
-
      <td
+
width="307">
-
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"
+
<p class="MsoNormal"
-
valign="top" width="307">
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-
      <p class="MsoNormal"
+
lang="EN-US">B</span></p>
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">B</p>
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
align="center"><img style="width: 242px; height: 220px;" id="Grafik 19"
-
align="center"><img id="Grafik 19"
+
src="http://2010.igem.org/wiki/images/f/fc/Freiburg10_Microscopy_Overlay_grey_1_GOI.png"
-
src="Freiburg10_Modularization_GOI_02_files/image024.jpg"
+
alt=""></p>
-
alt="Description: http://2010.igem.org/wiki/images/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"
+
</td>
-
height="220" width="242"></p>
+
</tr>
-
      </td>
+
<tr>
-
    </tr>
+
<td style="padding: 0cm 5.4pt; width: 250.7pt;" valign="top"
-
    <tr>
+
width="334">
-
      <td
+
<p class="MsoNormal"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 250.7pt;"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-
valign="top" width="334">
+
lang="EN-US">C</span></p>
-
      <p class="MsoNormal"
+
<p class="MsoNormal"
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">C</p>
+
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
      <p class="MsoNormal"
+
align="center"><img style="width: 258px; height: 195px;" id="Grafik 16"
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
src="http://2010.igem.org/wiki/images/4/48/Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png"
-
align="center"><img id="Grafik 16"
+
alt=""></p>
-
src="Freiburg10_Modularization_GOI_02_files/image025.jpg"
+
</td>
-
alt="Description: http://2010.igem.org/wiki/images/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+
<td style="padding: 0cm 5.4pt; width: 230.4pt;" valign="top"
-
height="195" width="258"></p>
+
width="307">
-
      </td>
+
<p class="MsoNormal"
-
      <td
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"
+
lang="EN-US">D</span></p>
-
valign="top" width="307">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">D</p>
+
align="center"><img style="width: 257px; height: 194px;" id="Grafik 17"
-
      <p class="MsoNormal"
+
src="http://2010.igem.org/wiki/images/0/0a/Freiburg10_Microscopy_phasecontrast_2_GOI.png"
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+
alt=""></p>
-
align="center"><img id="Grafik 17"
+
</td>
-
src="Freiburg10_Modularization_GOI_02_files/image026.jpg"
+
</tr>
-
alt="Description: http://2010.igem.org/wiki/images/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+
<tr>
-
height="194" width="257"></p>
+
<td colspan="2" style="padding: 0cm 5.4pt; width: 481.1pt;"
-
      </td>
+
valign="top" width="641">
-
    </tr>
+
<p class="MsoCaption" style="text-indent: 0cm;"><a
-
    <tr>
+
name="_Ref275784524"><span lang="EN-US">Figure </span></a><span
-
      <td colspan="2"
+
lang="EN-US">7</span><span lang="EN-US">: </span><span
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
style="font-weight: normal;" lang="EN-US">Fluorescence microscopy
-
valign="top" width="641">
+
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
-
name="_Ref275784524">Figure </a>6: <span
+
-
style="font-weight: normal;">Fluorescence microscopy
+
(Exciatation: 505nm, Emission: 536nm) was performed for detection of
(Exciatation: 505nm, Emission: 536nm) was performed for detection of
transduced cell expression mVenus. A:Cells detected in bright field
transduced cell expression mVenus. A:Cells detected in bright field
picture B: Detection of mVenus expression can be observed.</span></p>
picture B: Detection of mVenus expression can be observed.</span></p>
-
      </td>
+
</td>
-
    </tr>
+
</tr>
-
  </tbody>
+
</tbody>
</table>
</table>
-
<p class="MsoNormal">&nbsp;</p>
+
</div>
-
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span
+
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
lang="DE">Analysis</span><span lang="DE">
+
<h4><a name="_Toc275800685"></a><a name="_Toc275797958"><span
-
</span>of Target Cells by Flow Cytometry demonstrates GOI
+
lang="EN-US">Analysis
-
Expression</h4>
+
of Target Cells by Flow Cytometry demonstrates GOI Expression</span></a></h4>
-
<p class="MsoNormal">Characterizing the function of the
+
<p class="MsoNormal"><span lang="EN-US">Characterizing
-
hGH terminator, the <i>beta-globin</i>
+
the function of
-
intron and the complete plasmid, several approaches were conducted
+
the hGH
-
followed by
+
terminator, the <i>beta-globin</i> intron and the complete
-
analysis via flow cytometry. </p>
+
plasmid,
-
<h5>Influence of hGH terminator BioBrick on GOI Expression</h5>
+
several
-
<p class="MsoNormal">The iGEM team Freiburg provides the
+
approaches were conducted followed by analysis via flow cytometry. </span></p>
-
hGH plolyadenylation
+
<h5><a name="_Toc275800686"></a><a name="_Toc275797959"><span
-
sequence within the ‘Virus Construction Kit’ due to the fact that
+
lang="EN-US">Influence
-
almost every
+
of hGH terminator BioBrick on GOI Expression</span></a></h5>
-
eukaryotic mRNA is processed at their 3´ and 5´end except for histone
+
<p class="MsoNormal"><span lang="EN-US">The
-
mRNAs (Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved
+
iGEM team Freiburg provides
-
sequences. First, the
+
the hGH
-
polyadenylation signal “AATAAA” which is recognized by the multiprotein
+
plolyadenylation sequence within the ‘Virus Construction Kit’ due to
-
complex
+
the fact
-
and second the GT-rich region (downstream sequence element, DSE) which
+
that almost every eukaryotic mRNA is processed at their 3´ and 5´end
-
is
+
except for
-
located 30 nucleotides downstream of the cleavage site. The assembled
+
histone mRNAs </span><span lang="EN-US">(Millevoi
-
3´end-processing machinery cleaves the mRNA transcript immediately
+
et al. 2006)</span><span lang="EN-US">. Pre-mRNAs
-
after a
+
contain
-
CA-nucleotide therefore defining the cleavage site (Danckwardt et al.
+
two canonical conserved sequences. First, the polyadenylation signal
-
2008)<span style="font-size: 12pt; line-height: 200%;">. </span>Recombinant
+
“AATAAA”
-
vectorplasmids were engineered containing the inverted terminal repeats
+
which is recognized by the multiprotein complex and second the GT-rich
-
(ITRs),
+
region
-
a strong eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as
+
(downstream sequence element, DSE) which is located 30 nucleotides
-
gene of
+
downstream
-
interest with and without the hGH terminator signal. Transduction of
+
of the cleavage site. The assembled 3´end-processing machinery cleaves
-
HT1080
+
the mRNA
-
cells with constant volume of viral particles containing the
+
transcript immediately after a CA-nucleotide therefore defining the
-
vectorplasmids and
+
cleavage
-
measuring mVenus expression 24-hours post infection by flow cytometry
+
site </span><span lang="EN-US">(Danckwardt et al.
-
demonstrated that transgene expression of the constructs lacking the
+
2008)</span><span style="font-size: 12pt; line-height: 200%;"
-
hGH
+
lang="EN-US">. </span><span lang="EN-US">Recombinant
-
termination signal is significantly reduced as shown in Figure 7 and
+
vectorplasmids
-
Figure 8 confirming the expected results that hGH is essential for mRNA
+
were engineered containing the inverted terminal repeats (ITRs), a
-
processing.
+
strong
-
The iGEM team Freiburg_Bioware 2010 therefore suggests using the
+
eukaryotic promoter (CMV promoter: <a href = "http://partsregistry.org/Part:BBa_K404102" target="blank" > BBa_K404102</a>) and mVenus as gene of
-
provided hGH
+
interest
-
termination signal within the Virus Construction Kit for optimal gene
+
with and without the hGH terminator signal. Transduction of HT1080
-
expression.</p>
+
cells with constant
 +
volume of viral particles containing the vectorplasmids and measuring
 +
mVenus
 +
expression 24 hours post infection by flow cytometry demonstrated that
 +
transgene expression of the constructs lacking the hGH termination
 +
signal is
 +
significantly reduced as shown in </span><span lang="EN-US">Figure
 +
7</span><span lang="EN-US"> and </span><span lang="EN-US">Figure 8</span><span
 +
lang="EN-US">
 +
confirming the expected results
 +
that hGH is essential for mRNA processing. The iGEM team
 +
Freiburg_Bioware 2010
 +
therefore suggests using the provided hGH termination signal within the
 +
Virus
 +
Construction Kit for optimal gene expression.</span></p>
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
-
border="1" cellpadding="0" cellspacing="0">
+
border="0" cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr>
+
<tr>
-
      <td
+
<td
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+
style="padding: 0cm 5.4pt; vertical-align: top; width: 481.1pt;">
-
valign="top" width="641">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid
+
lang="EN-US">Vectorplasmid lacking hGH termination signal</span></b></p>
-
lacking hGH termination signal</b></p>
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
+
id="Grafik 30"
-
id="Grafik 30"
+
src="http://2010.igem.org/wiki/images/c/c7/Freiburg10_FACS_woHGH.png"
-
src="Freiburg10_Modularization_GOI_02_files/image027.gif"
+
height="408" width="629"></p>
-
height="408" width="629"></p>
+
</td>
-
      </td>
+
</tr>
-
    </tr>
+
<tr>
-
    <tr>
+
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
-
      <td
+
width="641">
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
<p class="MsoNormal"
-
valign="top" width="641">
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-
      <p class="MsoNormal"
+
lang="EN-US">Vectorplasmid containing hGH terminator signal</span></b></p>
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid
+
<p class="MsoNormal"
-
containing hGH terminator signal</b></p>
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-
      <p class="MsoNormal"
+
id="Grafik 2049"
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+
src="http://2010.igem.org/wiki/images/7/70/Freiburg10_FACS_withHGH.png"
-
id="Grafik 2049"
+
height="410" width="635"></p>
-
src="Freiburg10_Modularization_GOI_02_files/image028.gif"
+
</td>
-
height="410" width="636"></p>
+
</tr>
-
      </td>
+
<tr>
-
    </tr>
+
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
-
    <tr>
+
width="641">
-
      <td
+
<p class="MsoNormal"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a
-
valign="top" width="641">
+
name="_Ref275784539"><b><span lang="EN-US">Figure </span></b></a><b><span
-
      <p class="MsoNormal"
+
lang="EN-US">8</span></b><b><span lang="EN-US">:</span></b><span
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a
+
lang="EN-US"> <b>Flow cytometry analysis of vectorplasmids with and
-
name="_Ref275784539"><b>Figure </b></a><b>7</b><b>:</b>
+
without hGH terminator.</b> A: Gating non transduced cells
-
      <b>Flow cytometry analysis of vectorplasmids with and
+
(control);
-
without hGH terminator.</b> </p>
+
subcellular debris and clumps can be distinguished from single cells by
-
      <p class="MsoNormal"
+
size, estimated forward scatter (FS Lin) and granularity, estimated
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>A</b>:
+
side scatter (SS Lin) B: Non transduced cells applied against mVenus
-
Gating non transduced cells (control); subcellular debris and clumps
+
(Analytical gate was set such that 1% or fewer of negative control
-
can be distinguished from single cells by size, estimated forward
+
cells fell within the positive region (R5). C: Gating transduced cells
-
scatter (FS Lin) and granularity, estimated side scatter (SS Lin) <b>B</b>:
+
(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
-
Non transduced cells applied against mVenus (Analytical gate was set
+
lang="EN-US">≙</span><span lang="EN-US">R14)
-
such that 1% or fewer of negative control cells fell within the
+
(used plasmids for
-
positive region (R5). <b>C</b>: Gating transduced cells
+
transfection: GOI: pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR
-
(R2 <span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;">≙</span>R14)
+
(<a href = "http://partsregistry.org/Part:BBa_K404127" target="blank" > BBa_K404127</a>), pHelper, pRC). D: Transduced cells plotted against
-
(used plasmids for transfection: GOI: <b>pSB1C3_lITR_CMV_beta-globin
+
mVenus, R10 comprises transduced cells by detecting mVenus expression.
-
intron_mVenus_rITR (BBa_K404127)</b>, pHelper, pRC). <b>D</b>:
+
E: Overlay of non-transduced (red) and transduced (green) cells applied
-
Transduced cells plotted against mVenus, R10 comprises transduced cells
+
against mVenus.F: Gating non-transduced cells (control) G:
-
by detecting mVenus expression. <b>E</b>: Overlay of
+
Non-transduced cells applied against mVenus. H: Gating transduced cells
 +
(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
 +
lang="EN-US">≙</span><span lang="EN-US">R14)
 +
(used plasmids for
 +
transfection: GOI: reassembled
 +
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>), pHelper,
 +
pRC). I: Transduced cells applied against mVenus, R10 comprised
 +
transduced cells, by detecting mVenus expression. J: Overlay of
non-transduced (red) and transduced (green) cells applied against
non-transduced (red) and transduced (green) cells applied against
-
mVenus.<b>F</b>: Gating non-transduced cells (control) <b>G</b>:
+
mVenus.</span></p>
-
Non-transduced cells applied against mVenus. <b>H</b>:
+
</td>
-
Gating transduced cells (R2 <span
+
</tr>
-
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;">≙</span>R14)
+
</tbody>
-
(used plasmids for transfection: GOI: reassembled <b>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+
-
(BBa_K404119)</b>, pHelper, pRC). <b>I</b>:
+
-
Transduced cells applied against mVenus, R10 comprised transduced
+
-
cells, by detecting mVenus expression. <b>J</b>: Overlay
+
-
of non-transduced (red) and transduced (green) cells applied against
+
-
mVenus.</p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
</table>
</table>
-
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
+
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr>
+
<tr style="height: 319.2pt;">
-
      <td
+
<td style="padding: 0cm 5.4pt; width: 351.1pt; height: 319.2pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+
valign="top" width="468">
-
valign="top" width="641">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p>
+
lang="EN-US">&nbsp;</span></p>
-
      <p class="MsoNormal"
+
<p class="MsoNormal"
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-
      <img id="Diagramm 3"
+
align="center"><img id="Diagramm 3"
-
src="Freiburg10_Modularization_GOI_02_files/image029.gif"
+
src="http://2010.igem.org/wiki/images/9/94/Freiburg10_Diagram_hGH.png"
-
height="355" width="473"></p>
+
height="355" width="473"></p>
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
<p class="MsoCaption" style="text-indent: 0cm;"><a
-
name="_Ref275784545">Figure </a>8: Flow cytometry
+
name="_Ref275784545"><span lang="EN-US">Figure </span></a><span
-
analysis of vectorplasmids with and without hGH terminator.<span
+
lang="EN-US">9</span><span lang="EN-US">:
-
style="font-weight: normal;"> YFP expression of viral
+
Flow cytometry analysis of
-
genomes was determined by flow cytomery after 24-hour post infection.
+
vectorplasmids with and without hGH terminator.</span><span
 +
style="font-weight: normal;" lang="EN-US"> YFP
 +
expression of viral
 +
genomes was determined by flow cytomery 24 hour post infection.
Results demonstrate that mVenus expression of vectorplasmids lacking
Results demonstrate that mVenus expression of vectorplasmids lacking
the hGH terminator is reduced significantly proving that the
the hGH terminator is reduced significantly proving that the
Line 994: Line 740:
recombinant viral vectors engineered by using components of the Virus
recombinant viral vectors engineered by using components of the Virus
Construction Kit.</span></p>
Construction Kit.</span></p>
-
      </td>
+
</td>
-
    </tr>
+
</tr>
-
  </tbody>
+
</tbody>
</table>
</table>
-
<p class="MsoCaption">&nbsp;</p>
+
</div>
-
<h5>Influence of <i>Beta-globin</i> intron Biobrick
+
<p class="MsoCaption"><span lang="EN-US">&nbsp;</span></p>
-
on GOI Expression</h5>
+
<h5><a name="_Toc275800687"></a><a name="_Toc275797960"><span
-
<p class="MsoNormal">Providing an element assumed to be an
+
lang="EN-US">Influence
-
enhancer of transgene
+
of <i>Beta-globin</i> intron Biobrick on GOI Expression</span></a></h5>
-
expression (Nott et al. 2003), the iGEM team Freiburg tested a
+
<p class="MsoNormal"><span lang="EN-US">Providing
-
beta-globin
+
an element assumed to
-
intron derived from the human <i>beta globin</i> gene
+
be an
-
which can be fused
+
enhancer of transgene expression </span><span lang="EN-US">(Nott
-
upstream of the desired gene of interest. The beta-globin intron
+
et
-
BioBrick
+
al. 2003)</span><span lang="EN-US">, the iGEM team
-
consists of a partial chimeric CMV promoter followed by the intron II
+
Freiburg tested
-
of the <i>beta-globin</i>
+
a beta-globin intron derived from the human <i>beta globin</i>
-
gene. The 3´end of the intron is fused to the first 25 bases of human <i>beta
+
gene
-
globin</i> gene exon 3. The <i>beta globin</i>
+
which can
-
intron BioBrick is assumed to
+
be fused upstream of the desired gene of interest. The beta-globin
-
enhance eukaryotic gene expression (Nott et al. 2003). Analysis was
+
intron
-
conducted as described for the hGH terminator experiment
+
BioBrick consists of a partial chimeric CMV promoter followed by the
-
(see above). As shown in Figure 9 and Figure 10 the vectorplasmid
+
intron II
 +
of the <i>beta-globin</i> gene. The 3´end of the intron is
 +
fused to
 +
the first 25
 +
bases of human <i>beta globin</i> gene exon 3. The <i>beta
 +
globin</i>
 +
intron
 +
BioBrick is assumed to enhance eukaryotic gene expression </span><span
 +
lang="EN-US">(Nott et al. 2003)</span><span lang="EN-US">. Analysis
 +
was conducted as
 +
described for the hGH terminator experiment (see above). As shown in </span><span
 +
lang="EN-US">Figure 9</span><span lang="EN-US">
 +
and </span><span lang="EN-US">Figure 10</span><span lang="EN-US"> the
 +
vectorplasmid
missing the <i>beta-globin</i>
missing the <i>beta-globin</i>
intron showed a negligible difference in mVenus expression compared to
intron showed a negligible difference in mVenus expression compared to
viral
viral
genomes containing the <i>beta-globin</i> intron.
genomes containing the <i>beta-globin</i> intron.
-
Considering these results and
+
Considering these
 +
results and
taking into account that a constant volume of viral particles has been
taking into account that a constant volume of viral particles has been
used for
used for
Line 1,027: Line 787:
lacking the
lacking the
beta-globin intron is minimal. Since packaging efficiency of the AAV-2
beta-globin intron is minimal. Since packaging efficiency of the AAV-2
-
decreases with increasing sizes of the insert (Dong et al. 1996), the
+
decreases with increasing sizes of the insert </span><span lang="EN-US">(Dong
-
iGEM team Freiburg_Bioware suggests using the <i>beta-globin </i>intron
+
et al. 1996)</span><span lang="EN-US">, the iGEM
-
in dependence on the size of your transgene.</p>
+
team
 +
Freiburg_Bioware suggests using the <i>beta-globin </i>intron
 +
in
 +
dependence on
 +
the size of your transgene.</span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
+
style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
-
border="1" cellpadding="0" cellspacing="0"
+
border="0" cellpadding="0" cellspacing="0" width="654">
-
width="654">
+
<tbody>
-
  <tbody>
+
<tr style="height: 2.5pt;">
-
    <tr style="height: 2.5pt;">
+
<td style="padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;"
-
      <td
+
valign="top" width="654">
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;"
+
<p class="MsoNormal"
-
valign="top" width="654">
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-
      <p class="MsoNormal"
+
lang="EN-US">Vectorplasmid lacking <i>beta-globin</i>
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid
+
intron</span></b></p>
-
lacking <i>beta-globin</i> intron</b></p>
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
+
id="Grafik 55"
-
id="Grafik 55"
+
src="http://2010.igem.org/wiki/images/7/7c/Freiburg10_FACS_betaglobin.png"
-
src="Freiburg10_Modularization_GOI_02_files/image030.gif"
+
height="412" width="640"></p>
-
height="412" width="640"></p>
+
</td>
-
      </td>
+
</tr>
-
    </tr>
+
<tr style="height: 106.5pt;">
-
    <tr style="height: 106.5pt;">
+
<td style="padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
-
      <td
+
valign="top" width="654">
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
+
<p class="MsoNormal"
-
valign="top" width="654">
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-
      <p class="MsoNormal"
+
lang="EN-US">Vectorplasmid containing <i>beta-globin</i>
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid
+
intron</span></b></p>
-
containing <i>beta-globin</i> intron</b></p>
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+
id="Grafik 63"
-
id="Grafik 63"
+
src="http://2010.igem.org/wiki/images/8/83/Freiburg10_FACS_withbetaglobin.png"
-
src="Freiburg10_Modularization_GOI_02_files/image031.gif"
+
height="410" width="635"></p>
-
height="410" width="636"></p>
+
</td>
-
      </td>
+
</tr>
-
    </tr>
+
<tr style="height: 106.5pt;">
-
    <tr style="height: 106.5pt;">
+
<td style="padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
-
      <td
+
valign="top" width="654">
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
+
<p class="MsoCaption" style="text-indent: 0cm;"><a
-
valign="top" width="654">
+
name="_Ref275784803"><span lang="EN-US">Figure </span></a><span
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
lang="EN-US">10</span><span lang="EN-US">:
-
name="_Ref275784803">Figure </a>9: Flow cytometry
+
Flow cytometry analysis of
-
analysis of vectorplasmids with and without <i>beta-globin</i>
+
vectorplasmids with and without <i>beta-globin</i> intron.
-
intron.&nbsp; A<span style="font-weight: normal;">:
+
A</span><span style="font-weight: normal;" lang="EN-US">:
-
Gating non transduced cells (control); subcellular debris and clumps
+
Gating non transduced
-
can be distinguished from single cells by size, estimated forward
+
cells (control); subcellular debris and clumps can be distinguished
-
scatter (FS Lin) and granularity, estimated side scatter (SS Lin) </span>B<span
+
from single cells by size, estimated forward scatter (FS Lin) and
-
style="font-weight: normal;">: Non transduced cells applied
+
granularity, estimated side scatter (SS Lin) </span><span lang="EN-US">B</span><span
-
against mVenus (Analytical gate was set such that 1% or fewer of
+
style="font-weight: normal;" lang="EN-US">: Non
-
negative control cells fell within the positive region (R5). </span>C<span
+
transduced cells
-
style="font-weight: normal;">: Gating transduced cells (R2 </span><span
+
applied against mVenus (Analytical gate was set such that 1% or fewer
-
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span
+
of negative control cells fell within the positive region (R5). </span><span
-
style="font-weight: normal;">R14) (used plasmids for
+
lang="EN-US">C</span><span style="font-weight: normal;" lang="EN-US">:
-
transfection: GOI: </span>pSB1C3_lITR_CMV_mVenus_hGH_rITR
+
Gating transduced cells (R2 </span><span
-
(BBa_K404128)<span style="font-weight: normal;">, pHelper,
+
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
-
pRC). </span>D<span style="font-weight: normal;">:
+
lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
-
Transduced cells plotted against mVenus, R10 comprised transduced
+
(used plasmids for transfection: GOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_mVenus_hGH_rITR
-
cells, by detecting mVenus expression </span>E<span
+
(<a href = "http://partsregistry.org/Part:BBa_K404128" target="blank" > BBa_K404128</a>)</span><span style="font-weight: normal;" lang="EN-US">,
-
style="font-weight: normal;">: Overlay of non-transduced
+
pHelper, pRC). </span><span lang="EN-US">D</span><span
-
(red) and transduced (green) cells applied against mVenus </span>F<span
+
style="font-weight: normal;" lang="EN-US">:
-
style="font-weight: normal;">: Gating non-transduced cells
+
Transduced cells plotted
-
(control). </span>G<span style="font-weight: normal;">:
+
against mVenus, R10 comprised transduced cells, by detecting mVenus
-
Non-transduced cells applied against mVenus (R5).</span>H<span
+
expression </span><span lang="EN-US">E</span><span
-
style="font-weight: normal;">: Gating transduced cells (R2 </span><span
+
style="font-weight: normal;" lang="EN-US">: Overlay
-
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span
+
of non-transduced
-
style="font-weight: normal;">R14) (used plasmids for
+
(red) and transduced (green) cells applied against mVenus </span><span
-
transfection: GOI: reassembled </span>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+
lang="EN-US">F</span><span style="font-weight: normal;" lang="EN-US">:
-
(BBa_K404119)<span style="font-weight: normal;">, pHelper,
+
Gating non-transduced cells (control). </span><span lang="EN-US">G</span><span
-
pRC). </span>I<span style="font-weight: normal;">:
+
style="font-weight: normal;" lang="EN-US">:
 +
Non-transduced cells
 +
applied against mVenus (R5).</span><span lang="EN-US">H</span><span
 +
style="font-weight: normal;" lang="EN-US">: Gating
 +
transduced cells
 +
(R2 </span><span
 +
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
 +
lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
 +
(used plasmids for transfection: GOI: reassembled </span><span
 +
lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
 +
(<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>)</span><span style="font-weight: normal;" lang="EN-US">,
 +
pHelper, pRC). </span><span lang="EN-US">I</span><span
 +
style="font-weight: normal;" lang="EN-US">:
Transduced cells applied against mVenus, R10 comprised transduced
Transduced cells applied against mVenus, R10 comprised transduced
-
cells, by detecting mVenus expression. </span>J<span
+
cells, by detecting mVenus expression. </span><span lang="EN-US">J</span><span
-
style="font-weight: normal;">: Overlay of non-transduced
+
style="font-weight: normal;" lang="EN-US">: Overlay
 +
of non-transduced
(red) and transduced (green) cells applied against mVenus.</span></p>
(red) and transduced (green) cells applied against mVenus.</span></p>
-
      </td>
+
</td>
-
    </tr>
+
</tr>
-
  </tbody>
+
</tbody>
</table>
</table>
-
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
+
</div>
 +
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
+
style="border: medium none ; width: 306.65pt; border-collapse: collapse;"
-
border="1" cellpadding="0" cellspacing="0"
+
border="0" cellpadding="0" cellspacing="0" width="409">
-
width="654">
+
<tbody>
-
  <tbody>
+
<tr style="height: 87.3pt;">
-
    <tr style="height: 90.2pt;">
+
<td style="padding: 0cm 5.4pt; width: 306.65pt; height: 87.3pt;"
-
      <td
+
valign="top" width="409">
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 90.2pt;"
+
<p class="MsoNormal"
-
valign="top" width="654">
+
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-
      <p class="MsoNormal"
+
align="center"><img id="Diagramm 57"
-
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+
src="http://2010.igem.org/wiki/images/2/26/Freiburg10_Diagram_betaglobin.png.png"
-
align="left">&nbsp;</p>
+
height="332" width="450"></p>
-
      <p class="MsoNormal"
+
<p class="MsoCaption" style="text-indent: 0cm;"><a
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+
name="_Ref275784805"><span lang="EN-US">Figure </span></a><span
-
align="center"><img id="Diagramm 57"
+
lang="EN-US">11</span><span lang="EN-US">:
-
src="Freiburg10_Modularization_GOI_02_files/image032.gif"
+
Flow cytometry analysis of
-
height="332" width="450"></p>
+
vectorplasmids with and without <i>beta-globin</i> intron.</span><span
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
style="font-weight: normal;" lang="EN-US"> 48 hours
-
name="_Ref275784805">Figure </a>10: Flow cytometry
+
post transfection,
-
analysis of vectorplasmids with and without <i>beta-globin</i>
+
viral particles were harvested by freeze-thaw lysis and centrifugation
-
intron.<span style="font-weight: normal;"> 48-hours post
+
followed by HT1080 transduction. YFP expression of vectorplasmids was
-
transfection, viral particles were harvested by freeze-thaw lysis and
+
determined by flow cytometry 24 hours post infection. The vectorplasmid
-
centrifugation followed by HT1080 transduction. YFP expression of
+
missing the beta-globin intron showed a negligible difference in mVenus
-
vectorplasmids was determined by flow cytometry 24-hours post
+
expression compared to viral plasmid containing the beta-globin intron.</span></p>
-
infection. The vectorplasmid missing the beta-globin intron showed a
+
</td>
-
negligible difference in mVenus expression compared to viral plasmid
+
</tr>
-
containing the beta-globin intron.</span></p>
+
</tbody>
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
</table>
</table>
-
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p>
+
</div>
-
<h5>Functionality of the Full Assembled Vectorplasmid
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
Demonstrated by GOI Expression
+
<h5><a name="_Toc275800688"></a><a name="_Toc275797961"><span
-
</h5>
+
lang="EN-US">Functionality
-
<p class="MsoNormal">After assembly of plasmids containing
+
of the Full Assembled Vectorplasmid Demonstrated by GOI Expression</span></a><span
-
all required elements
+
lang="EN-US"> </span></h5>
-
(see Figure 1), functionality was tested in cell culture. AAV-293 cells
+
Producing recombinant virus particles for therapeutical applications
-
stably
+
is, besides specific cell targeting, purification and quantification
-
expressing E1A and E1B proteins were transfected with three plasmids
+
assays of AAV-2, one intention of the Virus Construction Kit provided
-
&nbsp;(pHelper,
+
by the iGEM team Freiburg_Bioware 2010. For obtaining a modular
-
pRC, pGOI). Virus particles were harvested 72-hours post-transfection
+
toolkit, the complex biological system of the Adeno-associated virus
-
and the
+
serotype 2 was examined by an exhaustive literature search.
-
tumor cell line HT1080 was transduced with the recombinant viral
+
Subsequently, the essential components for AAV-2 particle production
-
vectors
+
were extracted and redesigned to match the iGEM standard.<br>
-
encapsidating the gene of interest mVenus (BBa_I757008).</p>
+
The provided tripartite system is independent of a superinfection&nbsp;
-
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010
+
of Adeno- or herpes simplex viruses since the genes encoding the
-
compared the standard-plasmid
+
required helper-proteins are co-transfected. Inside the eukaryotic host
-
containing a subcloned mVenus (pAAV_mVenus, derived from the Stratagene
+
cell, the DNA sequence containing the inverted terminal repeats (ITRs)
-
system)
+
is extracted and later encapsidated into the preformed capsids after
-
with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+
production of single-stranded DNA. Consequently, this plasmid is known
-
(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by
+
as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH
 +
terminator signal are flanked by the ITRs (ITRs, <a href = "http://partsregistry.org/Part:BBa_K404100" target="blank" > BBa_K404100</a> and
 +
<a href = "http://partsregistry.org/Part:BBa_K404101" target="blank" > BBa_K404101</a>) and regulate transgene expression. The vector plasmid
 +
containing the desired gene of interest is cotransfected with the
 +
RepCap plasmid (<a href = "http://partsregistry.org/Part:BBa_K404001" target="blank" > BBa_K404001</a>, <a href = "http://partsregistry.org/Part:BBa_K404102" target="blank" > BBa_K404102</a> or <a href = "http://partsregistry.org/Part:BBa_K404103" target="blank" > BBa_K404103</a>) and the
 +
pHelper plasmid. To obtain the fully assembled vector plasmid, several
 +
assembly steps have to be performed. &nbsp;<br>
 +
After assembly of plasmids containing all required elements, vector
 +
plasmid functionality was confirmed in cell culture. AAV-293 cells
 +
stably expressing the E1A and E1B proteins were transfected with three
 +
plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72 hours
 +
post transfection and the tumor cell line HT1080 was transduced with
 +
the recombinant viral vectors encapsidating the gene of interest mVenus
 +
(<a href = "http://partsregistry.org/Part:BBa_I757008" target="blank" > BBa_I757008</a>).<br>
 +
In the beginning, the iGEM team Freiburg_Bioware 2010 used a commercial
 +
vector plasmid, pAAV-MCS (Stratagene), to determine whether virus
 +
particle production by AAV-293 cells could be achieved. iGEM RFC 25
 +
restriction enzyme sites were introduced and the fluorescent protein
 +
mVenus was subcloned into this plasmid. Subsequently, this plasmid was
 +
used as a reference and compared to the assembled vector plasmid
 +
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (abbreviated pSB1C3_mVenus,
 +
<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>). Fluorescence expression data obtained by flow cytometry
 +
analysis are shown in Figure 11 and Figure 12. Comparing mVenus
 +
expression of the reference plasmid and the pSB1C3_mVenus plasmid
 +
revealed that biological functionality of the reassembled plasmid was
 +
preserved. <br>
 +
<br>
 +
<div align="center">
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse;" border="0"
 +
cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
 +
width="641">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>pSB1C3_mVenus
 +
(<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>)</b></p>
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
 +
id="Grafik 2068"
 +
src="http://2010.igem.org/wiki/images/9/9e/Freiburg10_FACS_FULL_pSB1c3_mVenus.png"
 +
height="409" width="634"></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
 +
width="641">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
 +
lang="EN-US">pAAV_mVenus (reference)<br>
 +
</span></b></p>
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
 +
id="Grafik 89"
 +
src="http://2010.igem.org/wiki/images/a/ad/Freiburg10_FACS_FULL_pAAV_mVenus.png"
 +
height="407" width="630"></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
 +
width="641">
 +
<p class="MsoCaption" style="text-indent: 0cm;"><a
 +
name="_Ref275784576"><span lang="EN-US">Figure </span></a><span
 +
lang="EN-US">12</span><span lang="EN-US">: Flow cytometry analysis of
 +
fluorescent protein expression in transduced HT1080 cells. For viral
 +
particle production, AAV-293 cells were transfected with the
 +
reassembled vector plasmid (<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>) or the reference plasmid,
 +
respectively. A: Gating non transduced cells (control); subcellular
 +
debris and cellular aggreates can be distinguished from single cells by
 +
size, estimated forward scatter (FS Lin) and granularity, estimated
 +
side scatter (SS Lin) B: Non-transduced cells plotted against cells
 +
expressing mVenus (Analytical gate was set such that 1% or fewer of
 +
negative control cells fell within the positive region (R5) C: Gating
 +
transduced cells (R2 ≙ R14) (plasmids used for transfection: pGOI:
 +
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus:
 +
<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>), pHelper, pRC. D: Transduced cells plotted against cells
 +
expressing mVenus. R10 comprises transduced cells detected by mVenus
 +
fluorescence. E: Overlay of non-transduced (red) and transduced
 +
(green). F: Gating non-transduced cells (control). G: Non-transduced
 +
cells plotted against cells expressing mVenus (R5). H: Gating
 +
transduced cells (R14 ≙ R2) (plasmids used for transfection: pGOI:
 +
pAAV_mVenus, pHelper). I: Transduced cells plotted against cells
 +
expressing mVenus. R10 comprises transduced cells detected by mVenus
 +
fluorescence. J: Overlay of non-transduced (red) and transduced (green)
 +
cells plotted against mVenus expression.&nbsp;</span><span
 +
style="font-weight: normal;" lang="EN-US"> </span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
 +
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
 +
<div align="center">
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse;" border="0"
 +
cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
 +
width="641">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
lang="EN-US">&nbsp;</span></p>
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
 +
align="center"><img id="Diagramm 90"
 +
src="http://2010.igem.org/wiki/images/9/90/Freiburg10_Diagram_FULL_GOI.png"
 +
height="396" width="541"></p>
 +
<p class="MsoCaption" style="text-indent: 0cm;"><a
 +
name="_Ref275784852"><span lang="EN-US">Figure </span></a><span
 +
lang="EN-US">13</span><span lang="EN-US">: Quantification of mVenus
 +
fluorescence by flow cytometry analysis. The data source is identical
 +
to Figure 13. Compared to the reference vector plasmid, mVenus
 +
expression in HT1080 cells is enhanced in case of the assembled plasmid
 +
(pSB1C3_mVenus)&nbsp;</span><span style="font-weight: normal;"
 +
lang="EN-US"></span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800689"><span
 +
lang="EN-US"><span
 +
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
 +
lang="EN-US">Conclusion</span></a></h3>
 +
<p class="MsoNormal"><span lang="EN-US">Idea
 +
of the modular ‘Virus
 +
Construction
 +
Kit’ is to provide all required elements for producing recombinant,
 +
functional
 +
virus particles delivering encapsidated genes of interest to specific
 +
cells.
 +
First step was to modify and modularize the vectorplasmid comprising
 +
basically
 +
the cis-elements for replication (ITRs), a strong eukaryotic or tissue
 +
specific
 +
promoter (pCMV or phTERT), the gene of interest (fluorescent proteins
 +
or
 +
suicide genes) and the hGH termination signal. Each element was
 +
successfully
 +
cloned and reassembled resulting in functional vectorplasmids
 +
determined by flow
 +
cytometry and fluorescence microscopy analyses. Experiments have been
 +
performed
 +
with mVenus since measurement of fluorescent proteins can be easily
 +
performed
 +
and visualized. Considering the results, the iGEM team Freiburg_Bioware
 +
2010
 +
then tested the construct containing the suicide genes thymidine kinase
 +
and
 +
cytosine deaminase. Further details demonstrating efficient tumor
 +
killing,
 +
using prodrug-activating systems, see results page <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results#modularization">Arming</a>. </span></p>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800690"><span
 +
lang="EN-US"><span
 +
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
 +
lang="EN-US">References</span></a></h3>
 +
<p style="text-indent: 36pt;"><span
 +
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US">Danckwardt, S., Hentze, M.W. &amp; Kulozik,
 +
A.E., 2008. 3' end mRNA processing: molecular mechanisms and
 +
implications for
 +
health and disease. <i>The EMBO journal</i>, 27(3),
 +
482-98. Available
 +
at:
 +
http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p>
 +
<p style="text-indent: 36pt;"><span
 +
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US">Dong, J.Y., Fan, P.D. &amp; Frizzell, R.a.,
 +
1996. Quantitative analysis of the packaging capacity of recombinant
 +
adeno-associated virus. <i>Human gene therapy</i>, 7(17),
 +
2101-12.
 +
Available
 +
at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
 +
<p style="text-indent: 36pt;"><span
 +
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US">Millevoi, S. et al., 2006. An interaction
 +
between U2AF 65 and CF I(m) links the splicing and 3' end processing
 +
machineries. <i>The EMBO journal</i>, 25(20), 4854-64.
 +
Available at:
 +
http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p>
 +
<p style="text-indent: 36pt;"><span
 +
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US">Nott, A., Meislin, S.H. &amp; Moore, M.J.,
 +
2003. A quantitative analysis of intron effects on mammalian gene
 +
expression. <i>RNA
 +
(New York, N.Y.)</i>, 9(5), 607-17. Available at:
 +
http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
 +
<p style="text-indent: 36pt;"><span lang="EN-US">&nbsp;</span></p><br>
 +
 
 +
 
 +
 
 +
<a href = "http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Move to Top</a>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"
 +
style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;"
 +
align="left"><span lang="EN-US">&nbsp;</span></p>
 +
<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885921"></a><a
 +
name="_Toc275817880"><span lang="EN-US">Overview of RepVP123 plasmid</span></a></h2>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885922"></a><a
 +
name="_Toc275817881"><span lang="EN-US">Modularization: Overview</span></a></h3>
 +
<p class="MsoNormal"
 +
style="text-indent: 0cm; line-height: 150%; page-break-after: avoid;"><span
 +
lang="EN-US">In our terminology the term “RepVP123”
 +
encompasses the
 +
whole AAV2 genome excluding the ITRs. The <i>rep</i> locus
 +
comprises four
 +
proteins related to genome replication while the <i>cap</i>
 +
locus codes for the
 +
proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which
 +
are
 +
required for viral capsid assembly. Source of the RepVP123 BioBrick
 +
supplied
 +
within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the
 +
wild-type
 +
AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In
 +
order
 +
to introduce iGEM standard and additionally enabling the possibility to
 +
modify
 +
the viral capsid via integration of certain motives within the viral
 +
loops 453
 +
and 587 a total of twelve mutations within RepVP123 (see </span><span
 +
lang="EN-US">Figure 1</span><span lang="EN-US">)
 +
and additionally two mutations
 +
within the pSB1C3 backbone were performed either by Site-Directed
 +
Mutagenesis
 +
(SDM) or by ordering and cloning of specifically designed gene
 +
sequences matching
 +
the required demands. Modifying the pSB1C3 led to iGEM team
 +
Freiburg_Bioware’s
 +
variant of this backbone, pSB1C3_001.</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; width: 636px; height: 523px; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 462.5pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; line-height: 150%; page-break-after: avoid;"
 +
align="center"><img style="width: 605px; height: 451px;"
 +
id="Picture 17"
 +
src="http://2010.igem.org/wiki/images/0/0b/Freiburg10_pAAV_complete_mutations.png"
 +
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\RepCap_complete_modifications_arrows.jpg"></p>
 +
<p class="MsoCaption" style="text-indent: 0cm;"><a
 +
name="_Ref275820820"><span lang="EN-US">Figure </span></a><span
 +
lang="EN-US">1</span> <span
 +
style="font-size: 8pt; color: windowtext; font-weight: normal;"
 +
lang="EN-US">Mutations implemented into <i>RepVP123</i>
 +
in order to establish both iGEM standard and loop insertion capability.
 +
Green arrows indicate integrated restriction sites, red red arrows
 +
indicate deleted restriction sites. KpnI was deleted first and
 +
reinstated afterwards. (see text).</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<span
 +
style="font-size: 8pt; line-height: 150%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US"><br style="page-break-before: always;" clear="all">
 +
</span>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
style="font-size: 8pt; line-height: 150%;" lang="EN-US">&nbsp;</span></p>
 +
<table class="MsoNormalTable"
 +
style="border-collapse: collapse; width: 651px; height: 261px; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr style="height: 27pt;">
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 27pt;">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">Plasmid
 +
name:</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 27pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">Functionality
 +
(determinded in cell culture via transduction and flow cytometry ):</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 27pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">4x
 +
mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 27pt;"
 +
width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">inserted <i>rep</i>
 +
fragment</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 27pt;"
 +
width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">inserted<i> cap</i>
 +
fragment</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 27pt;"
 +
width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">reinstated KpnI</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 27pt;"
 +
width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">pAAV</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 27pt;"
 +
width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">pSB1C3_001</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 27pt;"
 +
width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">HSPG-ko</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC
 +
(wild-type)</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_4x
 +
mutant</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_inserts </span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">no</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_Cap</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_RepVP123</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 18pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 18pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_
 +
p5TATAless</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 18pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 18pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 27pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 27pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_
 +
HSPG-ko_p5TATAless</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 27pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 27pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoCaption" style="text-align: left;" align="left"><span
 +
lang="EN-US">Figure </span><span lang="EN-US">2</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Table
 +
contains complete overview about all plasmids containing <i>RepVP123</i>
 +
which
 +
were used by iGEM team Freiburg_Bioware 2010.</span></p>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885923"></a><a
 +
name="_Toc275817882"><span lang="EN-US">Modularization: Removing iGEM
 +
restriction
 +
sites and establishing loop insertion capability</span></a></h3>
 +
<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885924"></a><a
 +
name="_Toc275817883"><span lang="EN-US">Modifications in Rep</span></a></h4>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 576px; height: 191px;" id="Picture 4"
 +
src="http://2010.igem.org/wiki/images/b/bc/Freiburg10_rep_synthetic_gene_fragment.png"
 +
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg"></p>
 +
<p class="MsoCaption"><a name="_Ref275818017"></a><a
 +
name="_Ref275818042"><span lang="EN-US">Figure </span></a><span
 +
lang="EN-US">3</span> <span
 +
style="font-size: 8pt; color: windowtext; font-weight: normal;"
 +
lang="EN-US">Restriction sites within the wild-type <i>rep</i>
 +
gene sequence, which were removed via cloning of synthetized <i>rep </i>gene
 +
fragment into the plasmid. The red box indicates
 +
the region spanned by the synthetic sequence.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Making
 +
the <i>RepVP123</i>
 +
wild-type compatible with the iGEM standards required the removal of
 +
five
 +
restriction sites (see </span><span lang="EN-US">Figure
 +
1</span><span lang="EN-US">).
 +
This was achieved using site-directed mutagenesis for PstI (position
 +
310) and
 +
PstI (4073). The remaining three iGEM restriction sites EcoRI (1578),
 +
PstI
 +
(1773) and EcoRI (1796) were replaced by a synthetic gene fragment,
 +
since the <i>rep
 +
</i>ORF contained these restriction sites in close proximity to
 +
each other plus
 +
an additional KpnI restriction site which was also not desired (see </span><span
 +
lang="EN-US">Figure 2</span><span lang="EN-US">).
 +
This gene fragment was cloned into
 +
the <i>rep</i> gene using HindIII and SwaI, which are
 +
single-cutting
 +
restriction enzymes adjacent to the target area. Additionally, BamHI
 +
(859) and
 +
SalI (1239) were removed, because these enzymes were required for
 +
genetically
 +
inserting the loop modifications in VP123.</span></p>
 +
<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885925"></a><a
 +
name="_Toc275817884"><span lang="EN-US">Modifications in VP123</span></a></h4>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">In
 +
order to
 +
implement the restriction sites necessary for targeting via loop
 +
insertions,
 +
the gene coding for the VP proteins was modified as well. The
 +
introduction of
 +
these restriction required up to four base mutations in a row, hence it
 +
was
 +
decided to synthesize this gene fragment and replace the wild-type
 +
sequence in <i>RepVP123</i>
 +
as well.</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 574px; height: 218px;" id="Picture 5"
 +
src="http://2010.igem.org/wiki/images/c/cd/Freiburg10_cap_synthetic_gene_fragment.png"
 +
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg"></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">4</span> <span
 +
style="font-size: 8pt; color: windowtext; font-weight: normal;"
 +
lang="EN-US">Restriction sites within <i>cap</i>
 +
sequence showing introduced loop insertion restriction sites into <i>cap</i>
 +
to enable cloning of targeting or purification motifs into both 453 and
 +
587 loops. Again, the red box indicates gene sequence which was
 +
synthetized.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 511px; height: 302px;" alt=""
 +
id="Chart 7"
 +
src="http://2010.igem.org/wiki/images/6/6b/Freiburg10_wt_vs_4xmutant.png"></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">5</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Results
 +
for transduction efficiency measured by flow cytometry. Fluorescence is
 +
measured in surviving cells. The tested <i>RepVP123</i>
 +
containing four point mutation to delete iGEM and loop insertion
 +
restriction sites does not show any difference within mVenus expression
 +
compared to the wild-type and therefore can be verified working.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Alongside
 +
to
 +
creating an iGEM compatible plasmid, infectivity of the modified
 +
construct was
 +
tested in cell culture via flow cytometry. Then experiments confirmed
 +
that
 +
single cloning steps did not interfere with natural viral infectivity
 +
at first
 +
(see above). But cloning of the synthesized <i>rep</i>
 +
gene fragment into the
 +
plasmid dramatically reduced transduction efficiency as detected by
flow
flow
-
cytometry analysis are shown in Figure 11 and Figure 12. Comparing
+
cytometry. Scrutinizing each mutation and its potential impact,
-
mVenus expression
+
suggested that
-
of the standard plasmid and the modified, assembled plasmid reveals
+
abolished transduction was related to the mutation, which removed the
 +
KpnI
 +
(1721) site. This site is located within a splice site, which is
 +
crucial for
 +
the Rep proteins, and thus even silent mutations may interfere with
 +
virus
 +
production (see extra topic “Rep proteins”).</span></p>
 +
<span
 +
style="font-size: 11pt; line-height: 200%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US"><br style="page-break-before: always;" clear="all">
 +
</span>
 +
<p class="MsoNormal" style="text-indent: 0cm; text-align: center;"><span
 +
lang="EN-US">&nbsp;<img alt="xxx"
 +
src="http://2010.igem.org/wiki/images/f/fd/Freiburg10_Freiburg10_facs_table1.png"><br>
 +
</span></p>
 +
<br>
 +
<p style="text-align: center;" class="MsoNormal">&nbsp;<img
 +
style="width: 636px; height: 396px;" alt=""
 +
src="http://2010.igem.org/wiki/images/3/3a/Freiburg10_Freiburg10_facs_table2.png"></p>
 +
<br>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US"></span></p>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US"></span></p>
 +
<table
 +
style="width: 474px; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td style="width: 464px;">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 10pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
 +
align="center"><span style="font-size: 10pt;"><img
 +
style="width: 395px; height: 215px;" id="Chart 18"
 +
src="http://2010.igem.org/wiki/images/7/73/Freiburg10_KpnI_deleted.png"
 +
alt="Description: Fluorescence of HT1080 cells transduced with FRAGE AN ADRIAN:WELCHES GOI? and P325 and P326 both containing RepCap in which the synthetized gene sequences were inserted. As control,pAAV_RC (P50) containing wtRepCap was used. mVenus expression within cells clearly state that tested constructs does not meet expectations."></span></p>
 +
<p style="text-align: justify;" class="MsoCaption"><span
 +
lang="EN-US">Figure </span><span lang="EN-US">8</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">AAV-293
 +
cells were transfected with three plasmids pHelper,
 +
pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus _hGH_[AAV2]-right-ITR and
 +
pAAV_RC_inserts (see above) or pAAV_RC (see below) providing essential
 +
genes and proteins for producing viral particles. After 48 hour post
 +
transfection, viral particles were harvested by freeze-thaw lysis and
 +
centrifugation followed by HT1080 transduction. mVenus expression of
 +
viral genomes was determined by flow cytomery 24 hours post
 +
infection. </span><span style="color: windowtext; font-weight: normal;"
 +
lang="EN-US">Fluorescence
 +
is measured in surviving cells</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">.
 +
Results show that insertion of both <i>rep</i>
 +
and <i>cap</i> syntheses disrupts viral infectivity.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US"></span></p>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US">Therefore,
 +
additional constructs were designed containing only the synthetic <i>cap</i>
 +
sequence or both, the synthesized sequences plus a re-mutation of KpnI
 +
(1721), in
 +
order to re-establish the wild-type splice site within the <i>rep</i>
 +
ORF. Results
 +
from cell culture obtained via FACS revealed that in fact the poor
 +
results were
 +
related to the KpnI restriction site deletion. Both constructs showed a
 +
transduction efficiency corresponding to the unmodified wild-type <i>RepVP123</i>’s
 +
transduction efficiency.</span></p>
 +
<div align="center">
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse;" border="0"
 +
cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr style="height: 177.6pt;">
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 464.4pt; height: 177.6pt;">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 10pt; text-align: center; text-indent: 0cm; line-height: 115%; page-break-after: avoid;"
 +
align="center"><img style="width: 625px; height: 233px;" alt=""
 +
id="Chart 3"
 +
src="http://2010.igem.org/wiki/images/6/62/Freiburg10_KpnI_reinstated.png"></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">9</span> <span lang="EN-US">F</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">luorescence
 +
of cells transduced with mVenus carrying rAAV measured by flow
 +
cytometry. AAV-293 cells were transfected with three plasmids pHelper,
 +
pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR and <i>RepVP123</i>
 +
constructs providing essential genes and proteins for producing viral
 +
particles. 48 hours post transfection, viral particles were
 +
harvested by freeze-thaw lysis and centrifugation followed by HT1080
 +
transduction. mVenus expression of viral genomes was determined by flow
 +
cytometry 24 hours post infection. Results show that <i>cap </i>integration
 +
does not influence infectivity. </span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
 +
is measured in surviving cells.</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">
 +
Recreation
 +
of KpnI within <i>rep</i> splice site recovers
 +
transduction efficiency.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US">&nbsp;</span></p>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885926"></a><a
 +
name="_Toc275817885"><span lang="EN-US">Modularization: Adapting
 +
pSB1C3 to loop
 +
insertions – pSB1C3_001</span></a></h3>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US">To
 +
fulfill iGEM requirements all plasmids need to be submitted in pSB1C3,
 +
therefore primers were ordered for amplifying <i>RepVP123</i>
 +
containing all
 +
modifications done so far by PCR and cloning the into pSB1C3. Still,
 +
pSB1C3
 +
contains two restriction sites for SspI and PvuII restriction enzymes
 +
in its
 +
CAT marker. Since these are necessary for cloning ViralBricks in this
 +
vector,
 +
the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM
 +
Headquarters
 +
to implement a new standard for the pSB1C3 backbone which was named
 +
pSB1C3_001.
 +
Both restriction sites interfering with ViralBrick insertions were
 +
mutated to
 +
make SspI and PvuII single-cutters (see method development).</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; margin-left: auto; border-collapse: collapse; text-align: left; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 584px; height: 278px;"
 +
id="Picture 16"
 +
src="http://2010.igem.org/wiki/images/d/d1/Freiburg10_pSB1C3_001_mutations.png"
 +
alt=""></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">10</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Comparison
 +
of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and
 +
PvuII are marked by red boxes.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal"><i><span lang="EN-US">RepVP123</span></i><span
 +
lang="EN-US">
 +
containing both <i>rep</i> and <i>cap</i>
 +
synthetic gene fragments including
 +
the re-mutation of KpnI and the downstream p5TATA-less promotor was
 +
cloned into
 +
the newly constructed pSB1C3_001. Testing this newly assembled plasmid
 +
in cell
 +
culture revealed unexpected data. Not only did the newly assembled
 +
plasmid work
 +
(see Figure 10), but in comparison to pAAV containing the same <i>RepVP123</i>
 +
construct, pSB1C3_001 showed an about 3 times higher transduction
 +
efficiency.
 +
Although exact reasons are still unknown, these results are probably
 +
related to
 +
the reduced length of pSB1C3_001 compared to the original pAAV plasmid
 +
of
 +
approximately 1000 base pairs.</span></p>
 +
<span
 +
style="font-size: 11pt; line-height: 200%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US"></span><span lang="EN-US">&nbsp;</span>
 +
<table
 +
style="border: medium none ; width: 466px; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
class="MsoTableGrid" border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr style="height: 209.85pt;">
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 4661px; height: 209.85pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><span style="font-size: 10pt; line-height: 200%;"><img
 +
style="width: 516px; height: 258px;" alt="" id="Chart 15"
 +
src="http://2010.igem.org/wiki/images/2/2b/Freiburg10_pAAV_pSB1C3_001.png"></span></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">11</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">AAV-293
 +
cells were transfected with three plasmids pHelper,
 +
pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and
 +
pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR
 +
providing essential genes and proteins for producing viral particles.
 +
After 48 hours post transfection, viral particles were harvested by
 +
freeze-thaw lysis and centrifugation followed by HT1080 transduction.
 +
mVenus expression of viral genomes was determined by flow cytomery
 +
after 24 hours post infection. </span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
 +
is measured in surviving cells.</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">&nbsp;Results
 +
showed functionality of <i>RepVP123</i>
 +
within pSB1C3_001 vector and additionally increased transduction
 +
efficiency.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885927"></a><a
 +
name="_Toc275817886"><span lang="EN-US">Turning-off natural tropism:
 +
HSPG-knock-out</span></a></h4>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Shutting-down
 +
the
 +
natural viral tropism is essential for targeting specifically tumor
 +
cells and
 +
not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware
 +
2010
 +
decided to knock-out the viral natural tropism delivered by the heperan
 +
sulfate
 +
proteoglycan-(HSPG) binding site within the viruses 587 loop. The
 +
knock-out was
 +
cloned by designing primers containing the required base exchanges and
 +
performing a SDM. Like performed before, this <i>RepVP123</i>
 +
variant was tested
 +
in cell culture as well and evaluated by flow cytometry. Results show
that
that
-
biological functionality of the reassembled plasmid was confirmed. </p>
+
mutation of HSPG-binding motif has severe impact on transduction
 +
efficiency
 +
thus enabling a viral particle carrying this knock-out and additional
 +
targeting
 +
motifs, e.g. within the loops or presented via N-terminal fusion to
 +
bind target
 +
cells’ receptors and therefore infecting target cells at a much higher
 +
rate
 +
compared to unspecific infection of other cell types within an organism
 +
(see </span><span lang="EN-US">Figure 12</span><span lang="EN-US">).</span></p>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">To
 +
quantify
 +
differences in infectivity, the infectious titer of viral particles
 +
built-up of
 +
<i>RepVP123</i> with and without HSPG binding motif was
 +
determined by qPCR (see
 +
</span><span lang="EN-US">Figure 14</span><span lang="EN-US">) for
 +
different cell
 +
lines. Results show that the implemented HSPG-knock-out verifies
 +
results obtained
 +
from flow cytometry, infectious titers severely compared to <i>RepVP123</i>
 +
with intact HSPG binding motif.</span></p>
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
-
border="1" cellpadding="0" cellspacing="0">
+
border="0" cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr>
+
<tr style="height: 258.85pt;">
-
      <td
+
<td
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 459.85pt; height: 258.85pt;">
-
valign="top" width="641">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="text-indent: 0cm; page-break-after: avoid;"><img
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
+
style="width: 595px; height: 379px;" id="Picture 2"
-
lang="DE">pSB1C3_mVenus (BBa_K404119)</span></b></p>
+
src="http://2010.igem.org/wiki/images/9/9d/Freiburg10_HSPG-ko_sequence.png"
-
      <p class="MsoNormal"
+
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\HSPG-ko_modified.jpg"></p>
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
+
<p class="MsoCaption" style="text-align: left;" align="left"><span
-
id="Grafik 2068"
+
lang="EN-US">Figure </span><span lang="EN-US">12</span> <span
-
src="Freiburg10_Modularization_GOI_02_files/image033.gif"
+
style="color: windowtext; font-weight: normal;" lang="EN-US">Alignment
-
height="409" width="634"></p>
+
of 587 loop within viral VP123: The upper sequence shows a strand
-
      </td>
+
containing the HSPG binding motif (AGA, in red boxes), the lower
-
    </tr>
+
sequence contains the HSPG-ko introduced by the iGEM team
-
    <tr>
+
Freiburg_Bioware 2010 (GCT and GCC, blue boxes).</span></p>
-
      <td
+
</td>
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
</tr>
-
valign="top" width="641">
+
</tbody>
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>pAAV_mVenus
+
-
(Stratagene)</b></p>
+
-
      <p class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+
-
id="Grafik 89"
+
-
src="Freiburg10_Modularization_GOI_02_files/image034.gif"
+
-
height="406" width="630"></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
-
valign="top" width="641">
+
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
-
name="_Ref275784576">Figure </a>11: Flow cytometry
+
-
analysis of reassembled vectorplasmid (BBa_K404119) compared to
+
-
standard plasmid provided by Stratagene. A<span
+
-
style="font-weight: normal;">: Gating non transduced cells
+
-
(control); subcellular debris and clumps can be distinguished from
+
-
single cells by size, estimated forward scatter (FS Lin) and
+
-
granularity, estimated side scatter (SS Lin) B: Non transduced cells
+
-
plotted against mVenus (Analytical gate was set such that 1% or fewer
+
-
of negative control cells fell within the positive region (R5).C:
+
-
Gating transduced cells (R2 </span><span
+
-
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span
+
-
style="font-weight: normal;">R14) (used plasmids for
+
-
transfection: pGOI: </span>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+
-
(pSB1C3_mVenus: BBa_K404119<span
+
-
style="color: rgb(0, 176, 80); font-weight: normal;">)</span><span
+
-
style="font-weight: normal;">, pHelper, pRC. </span>D<span
+
-
style="font-weight: normal;">: Transduced cells plotted
+
-
against mVenus, R10 comprised transduced cells, by detecting mVenus
+
-
expression. </span>E<span style="font-weight: normal;">:
+
-
Overlay of non-transduced (red) and transduced (green). </span>F<span
+
-
style="font-weight: normal;">: Gating non transduced cells
+
-
(control). </span>G<span style="font-weight: normal;">:
+
-
Non-transduced cells plotted against mVenus (R5). </span>H<span
+
-
style="font-weight: normal;">: Gating transduced cells (R14 </span><span
+
-
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span
+
-
style="font-weight: normal;">R2) (used plasmids for
+
-
transfection: pGOI: pAAV_mVenus, pHelper). </span>I<span
+
-
style="font-weight: normal;">: Transduced cells plotted
+
-
against mVenus, R10 comprised transduced cells, by detecting mVenus
+
-
expression.</span> J<span style="font-weight: normal;">:
+
-
Overlay of non-transduced (red) and transduced (green) cells plotted
+
-
against mVenus expression. </span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
</table>
</table>
-
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
+
-
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
+
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;"
+
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
-
border="1" cellpadding="0" cellspacing="0">
+
border="0" cellpadding="0" cellspacing="0">
-
  <tbody>
+
<tbody>
-
    <tr>
+
<tr>
-
      <td
+
<td
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+
style="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
-
valign="top" width="641">
+
<p class="MsoNormal"
-
      <p class="MsoNormal"
+
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p>
+
align="center"><img style="width: 467px; height: 229px;" alt=""
-
      <p class="MsoNormal"
+
id="Chart 9"
-
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+
src="http://2010.igem.org/wiki/images/0/01/Freiburg10_columns_HSPG_HSPG_ko.png"></p>
-
align="center"><img id="Diagramm 90"
+
<p class="MsoCaption"><a name="_Ref275882130"><span lang="EN-US">Figure
-
src="Freiburg10_Modularization_GOI_02_files/image035.gif"
+
</span></a><span lang="EN-US">13</span> <span
-
height="396" width="541"></p>
+
style="color: windowtext; font-weight: normal;" lang="EN-US">Transduction
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
efficiency of HT1080 cells measured by flow cytometry.&nbsp;</span><span
-
name="_Ref275784852">Figure </a>12: Flow cytometry
+
style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
-
analysis of reassembled vectorplasmid (BBa_K404119) compared to
+
is measured in surviving cells.</span><span
-
standard plasmid provided by Stratagene. <span
+
style="color: windowtext; font-weight: normal;" lang="EN-US">
-
style="font-weight: normal;">Fluorescence of the standard
+
Knock-out of
-
plasmid pAAV_mVenus (Stratagene) and the recombinant pSB1C3_mVenus
+
HSPG binding motif greatly reduces transduction efficiency compared to <i>RepVP123</i>
-
(BBa_K404119) construct was measured. As demonstrated mVenus expression
+
containing the motiv.</span></p>
-
is enhanced in the assembled plasmid (pSB1C3_mVenus) compared to the
+
</td>
-
standard pAAV_mVenus construct.</span></p>
+
</tr>
-
      </td>
+
</tbody>
-
    </tr>
+
-
  </tbody>
+
</table>
</table>
-
<p class="MsoNormal">&nbsp;</p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<p style="margin-left: 24pt; text-indent: -24pt;"><span
+
<table class="MsoTableGrid"
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
-
lang="DE">Danckwardt, S.,
+
border="0" cellpadding="0" cellspacing="0">
-
Hentze, M.W. &amp; Kulozik, A.E., 2008. 3’ end mRNA processing:
+
<tbody>
-
molecular
+
<tr>
-
mechanisms and implications for health and disease. <i>The EMBO
+
<td
-
journal</i>,
+
style="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
-
27(3), pp.482-98. Available at:
+
<p class="MsoCaption"
-
http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p>
+
style="text-align: center; page-break-after: avoid;" align="center"><img
-
<p style="margin-left: 24pt; text-indent: -24pt;"><span
+
style="width: 535px; height: 456px;" alt="" id="Chart 1"
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
src="http://2010.igem.org/wiki/images/b/bc/Freiburg10_qPCR_HSPG_HSPG_ko.png"></p>
-
lang="DE">Dong, J.Y., Fan,
+
<p class="MsoCaption" style="text-align: center;" align="center"><a
-
P.D. &amp; Frizzell, R.A., 1996. Quantitative analysis of the
+
name="_Ref275883877"><span lang="EN-US">Figure </span></a><span
-
packaging
+
lang="EN-US">14</span> <span
-
capacity of recombinant adeno-associated virus. <i>Human gene
+
style="color: windowtext; font-weight: normal;" lang="EN-US">Infectious
-
therapy</i>,
+
titers of <i>RepVP123</i> with and without natural HSPG
-
7(17), pp.2101-12. Available at:
+
binding motif tested in different cell lines via qPCR. Shutting-down
-
http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
+
the HSPG binding motif reduces infectious titer in both HT1080 and HeLa
-
<p style="margin-left: 24pt; text-indent: -24pt;"><span
+
cell lines. For A431 cells, no infectious titer could be detected via
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
qPCR, which is probably related to poor transduction efficiency of A431
-
lang="DE">Millevoi, S. et
+
cells.</span></p>
-
al., 2006. An interaction between U2AF 65 and CF I(m) links the
+
</td>
-
splicing and 3’
+
</tr>
-
end processing machineries. <i>The EMBO journal</i>,
+
</tbody>
-
25(20), pp.4854-64.
+
</table>
-
Available at: http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p>
+
<a href = "http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Move to Top</a>
-
<p style="margin-left: 24pt; text-indent: -24pt;"><span
+
</div>
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
<br>
-
lang="DE">Nott, A., Meislin,
+
<br>
-
S.H. &amp; Moore, M.J., 2003. A quantitative analysis of intron
+
<br>
-
effects on
+
-
mammalian gene expression. <i>RNA (New York, N.Y.)</i>,
+
-
9(5), pp.607-17.
+
-
Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
+
-
<p style="margin-left: 24pt; text-indent: -24pt;">&nbsp;</p>
+
</html>
</html>

Latest revision as of 02:45, 28 October 2010

Modularization


Contents

Modularization of the vectorplasmid

Introduction to Modularization of Vectorplasmid. 1

Recombinant and Modular Vectorplasmid Carrying GOI 2

Cloning and Combination Strategies for the Vectorplasmid. 3

Testing functionality of Assembled Vectorplasmid. 7

Fluorescence Microscopy of Target Cells Demonstrates GOI Expression. 7

Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression. 8

Influence of hGH terminator BioBrick on GOI Expression. 8

Influence of Beta-globin intron Biobrick on GOI Expression. 11

Functionality of the Full Assembled Vectorplasmid Demonstrated by GOI Expression. 14

Conclusion. 16

References. 17

 

Modularization of the RepVP123 plasmids

Overview of RepVP123 plasmid. 2

Modularization: Overview.. 2

Modularization: Removing iGEM restriction sites and establishing loop insertion capability. 3

Modifications in Rep. 3

Modifications in VP123. 4

Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001. 7

Turning-off natural tropism: HSPG-knock-out. 9

 


Introduction to Modularization of vector plasmid


Producing recombinant virus particles for therapeutical means is, besides specifically target cells, purification and quantification assays of AAV-2, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex components of AAV-2 were extracted and redesigned to match the iGEM standard. Functional activity was tested in cell culture.

Differing from the wildtype AAV-2 genome, the Helper Free System provided by Stratagene comprises three plasmids and a specialized production cell line. AAV-293 cells derived from the HEK cell line express the stably integrated E1A and E1B helper proteins for efficient virus production. The plasmid containing the inverted terminal repeats (ITRs) is encapsidated into the preformed capsids after production of single-stranded DNA therefore also known as vectorplasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs and serve in the host cell for regulation of transgene expression. In addition to that, the plasmid coding for the Rep and Cap proteins (pRC) can be provided in trans leading to a layer of specificity due to the fact that the two genes are not packaged into the capsid since lacking of the ITRs impairs encapsidation. Another advantage of the Helper Free System can be attributed to cotransfection of another helper plasmid (pHelper), which provides the necessary proteins normally obtained by superinfection with helper viruses such as adenovirus or herpes simplex virus. These helper genes are required for full viral assembly by regulating gene expression of Rep and Cap proteins.

Recombinant and Modular Vectorplasmid Carrying GOI

The iGEM team Freiburg_Bioware 2010 provides a modular Virus Construction Kit for therapeutical applications, quantification assays and purification approaches depending on capsid modifications and the gene of interest flanked by the inverted terminal repeats (ITRs. In order to produce BioBrick-compatible standardized biological parts, we reengineered the plasmids and added new components for gene therapy approaches and analysis of biological activity of assembled BioBrick parts. Each element required for intact and functional plasmids comprising the ITRs, a promoter, a putative enhancer element and the hGH terminator was PCR amplified and fused together de novo. As shown in Figure 1, the vectorplasmid was assembled with the produced BioBricks consisting of the left and right ITR ( BBa_K404100 and BBa_K40410), a promoter (pCMV : BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin intron ( BBa_K404107), the gene of interests (fluorescent proteins mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30: ( BBa_K404112, mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator ( BBa_K404108).

Figure 1: Overview of the theoretical sequence of each BioBrick provided within the Virus Construction Kit for an intact and fully functional rAAV genome. The plasmid in the lowest panel was used for tumor killing in combination with plasmids coding for modified capsid proteins. More detailed infomartion about these constructs can be found under ‘Arming: Killing the tumor’ and ‘N-terminal fusion for Targeting’.

 

Cloning and Combination Strategies for the Vectorplasmid

Organization of the recombinant viral DNA was modified ensuring several layers of specificity to our systems including a tumor-specific promoter and suicide genes encoding prodrug convertases. In order to modularize the rAAV sequence, each plasmid element (Figure 1) was PCR-amplified and cloned into the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team Freiburg_Bioware 2010 performed three site-directed mutagenesis in the gene of interest TK30 ( BBa_K404109) and cytosine deaminase ( BBa_K404112) for deletion of PstI and NgoMIV iGEM site (for further information see the results page of ‘Arming – Killing the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich regions forming T-shaped hairpins during replication, PCR amplification was not possible. Hence a cloning strategy was developed by the iGEM team Freiburg in order to provide BioBrick-compatible ITRs (see ‘Method Development of Cloning Strategy for ITRs’).

In Figure 2 the schematic overview of the modularization process can be seen which has been followed to conduct the assembly steps required for functional vectorplasmids.

Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png

Figure 3: Assembly procedure for fusion of BioBricks and composite parts to a fully assembled and functional plasmid coding for your gene of interest. This plasmid can be cotransfected with two helper plasmids providing protein for assembly and encapsidating of the rAAV genome (your gene of interest) into the capsids.

 

The iGEM team Freiburg_Bioware provides two examples demonstrating the assembly procedure for constructing vectorplasmids. The first representative example is the fusion of the BioBrick part beta-globin to the composite parts containing the 5´ elements of the plasmids, which are left ITR and CMV or phTERT promoter, respectively.

As shown in Figure 3 the theoretical cloning performed for assembling the BioBricks beta-globin intron and leftITR_CMV together can be observed.

Figure 4: Theoretical cloning of the composite part leftITR_CMV to the beta-globin intron BioBrick leading to the plasmid leftITR_CMV_beta-globin intron.

 

The plasmids were digested with both XbaI and PstI (beta-globin intron: BBa_K404107) or SpeI and PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the preparative gel in Figure 4, the expected bands could be detected under UV light and the extracted DNA could be successfully ligated. Each assembly step for producing BioBrick intermediates was conducted following the same strategy.

 

Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png

Figure 5: Assembly intermediate in fusion of the vectorplasmids containing different promoters. Fusion of the BioBrick part beta-globin ( BBa_K404107) intron to the composite parts leftITR_pCMV and leftITR_phTERT, respectively, was performed following the BioBrick assembly strategy by digesting the insert with PstI and XbaI and the vectors with SpeI and PstI. The left lane shows the expected fragment at around 560 bp which corresponds to the beta-globin intron fragment, in contrast to the two lanes in the center and on the right which correspond to linearized plasmids after digesting with above mentioned iGEM restriction sites. M, GeneRuler DNA ladder mix; Insert, pSB1C3_beta-globin intron; Vector pCMV, pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.

 

Separated fragments were extracted using the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated with T4-ligase. After ligation has been carried out, E. coli XL-1B cells were transformed and incubated over night at 37°C. Picking clones from the transformation plate was performed the following day and DYT medium was inoculated incubating overnight. Plasmid DNA was isolated and test digestion revealed that cloning was successful obtaining the composite part leftITR_CMV_beta-globin intron ( BBa_K404117).

Plasmid production incorporating all required elements for transgene expression and genome encapsidation into empty viral capsids was performed by fusing the downstream elements consisting of the hGH terminator and right ITR to the intermediate part providing the gene of interest and the promoter fused to the left ITR. Figure 5 demonstrates the assembly performed with pSB1C3_leftITR_phTERT_beta-globin intron_mVenus and pSB1C3_hGH_rightITR ( BBa_K404116). The fragment obtained after digestion on the left lane fits to the hGH-terminator_rightITR length. The isolated fragments were ligated and successful assembly was confirmed by test digestion obtaining the vectorplasmid pSB1C3_leftITR_phTERT_beta-globin intron_mVenus_hGH_rightITR ( BBa_K404124).

Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png

Figure 6: Modularization of the assembled vectorplasmid containing the phTERT promoter and mVenus as gene of interest. Fusion of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part to the composite parts pSB1C3_hGH_rightITR was performed following the BioBrick assembly strategy by digesting the insert with XbaI and PstI and the vector with SpeI and PstI. The left lane corresponds to linearized plasmid after digesting with above mentioned iGEM restriction sites whereas the right lane reveals an intensive band at around 650 bp confirming the expected size of 657 bp of hGH_rITR. M, GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.

 

Since cloning does not confirm biological activity, we analyzed the plasmids and their functional components, hGH terminator and beta-globin intron, in cell culture. Assembled plasmids have been cotransfected, using AAV-293 cells, which provide the stable integrated E1A and E1B genes, with helper plasmids required for capsid assembly and genome encapsidation (pRC and pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus particles containing the single stranded DNA were harvested 72 hours post transfection and HT1080 cells transduced with constant volumes of viral vectors. 48 hours post infection; transduced cells expressing the gene of interest were analyzed by flow cytometry. Facilitating and demonstrating the analysis of functionality of the assembled plasmid, mVenus was used in first place since fluorescent proteins enable facile visualization using fluorescent microscopy and flow cytometry analysis.

Testing functionality of Assembled Vectorplasmid

Fluorescence Microscopy of Target Cells Demonstrates GOI Expression

Qualitative analysis of mVenus expression by fluorescence microscopy was conducted using Axio Observer Z1 showing that transduced HT1080 cells and non-transduced cells could be easily distinguished. In Figure 6 cells were excited with 505nm and fluorescence emission at 536nm was detected. Therefore, successful infection of tumor cells by recombinant viral particles carrying the assembled vectorplasmid coding for mVenus could be demonstrated.

A

B

C

D

Figure 7: Fluorescence microscopy (Exciatation: 505nm, Emission: 536nm) was performed for detection of transduced cell expression mVenus. A:Cells detected in bright field picture B: Detection of mVenus expression can be observed.

 

Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression

Characterizing the function of the hGH terminator, the beta-globin intron and the complete plasmid, several approaches were conducted followed by analysis via flow cytometry.

Influence of hGH terminator BioBrick on GOI Expression

The iGEM team Freiburg provides the hGH plolyadenylation sequence within the ‘Virus Construction Kit’ due to the fact that almost every eukaryotic mRNA is processed at their 3´ and 5´end except for histone mRNAs (Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved sequences. First, the polyadenylation signal “AATAAA” which is recognized by the multiprotein complex and second the GT-rich region (downstream sequence element, DSE) which is located 30 nucleotides downstream of the cleavage site. The assembled 3´end-processing machinery cleaves the mRNA transcript immediately after a CA-nucleotide therefore defining the cleavage site (Danckwardt et al. 2008). Recombinant vectorplasmids were engineered containing the inverted terminal repeats (ITRs), a strong eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of interest with and without the hGH terminator signal. Transduction of HT1080 cells with constant volume of viral particles containing the vectorplasmids and measuring mVenus expression 24 hours post infection by flow cytometry demonstrated that transgene expression of the constructs lacking the hGH termination signal is significantly reduced as shown in Figure 7 and Figure 8 confirming the expected results that hGH is essential for mRNA processing. The iGEM team Freiburg_Bioware 2010 therefore suggests using the provided hGH termination signal within the Virus Construction Kit for optimal gene expression.

Vectorplasmid lacking hGH termination signal

Vectorplasmid containing hGH terminator signal

Figure 8: Flow cytometry analysis of vectorplasmids with and without hGH terminator. A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells applied against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5). C: Gating transduced cells (R2 R14) (used plasmids for transfection: GOI: pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR ( BBa_K404127), pHelper, pRC). D: Transduced cells plotted against mVenus, R10 comprises transduced cells by detecting mVenus expression. E: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.F: Gating non-transduced cells (control) G: Non-transduced cells applied against mVenus. H: Gating transduced cells (R2 R14) (used plasmids for transfection: GOI: reassembled pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR ( BBa_K404119), pHelper, pRC). I: Transduced cells applied against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.

 

 

Figure 9: Flow cytometry analysis of vectorplasmids with and without hGH terminator. YFP expression of viral genomes was determined by flow cytomery 24 hour post infection. Results demonstrate that mVenus expression of vectorplasmids lacking the hGH terminator is reduced significantly proving that the polyadenylation signal is essential for viral gene expression using recombinant viral vectors engineered by using components of the Virus Construction Kit.

 

Influence of Beta-globin intron Biobrick on GOI Expression

Providing an element assumed to be an enhancer of transgene expression (Nott et al. 2003), the iGEM team Freiburg tested a beta-globin intron derived from the human beta globin gene which can be fused upstream of the desired gene of interest. The beta-globin intron BioBrick consists of a partial chimeric CMV promoter followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 25 bases of human beta globin gene exon 3. The beta globin intron BioBrick is assumed to enhance eukaryotic gene expression (Nott et al. 2003). Analysis was conducted as described for the hGH terminator experiment (see above). As shown in Figure 9 and Figure 10 the vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results and taking into account that a constant volume of viral particles has been used for transduction, the difference between the construct containing and lacking the beta-globin intron is minimal. Since packaging efficiency of the AAV-2 decreases with increasing sizes of the insert (Dong et al. 1996), the iGEM team Freiburg_Bioware suggests using the beta-globin intron in dependence on the size of your transgene.

Vectorplasmid lacking beta-globin intron

Vectorplasmid containing beta-globin intron

Figure 10: Flow cytometry analysis of vectorplasmids with and without beta-globin intron. A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells applied against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5). C: Gating transduced cells (R2 R14) (used plasmids for transfection: GOI: pSB1C3_lITR_CMV_mVenus_hGH_rITR ( BBa_K404128), pHelper, pRC). D: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression E: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus F: Gating non-transduced cells (control). G: Non-transduced cells applied against mVenus (R5).H: Gating transduced cells (R2 R14) (used plasmids for transfection: GOI: reassembled pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR ( BBa_K404119), pHelper, pRC). I: Transduced cells applied against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.

 

Figure 11: Flow cytometry analysis of vectorplasmids with and without beta-globin intron. 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. YFP expression of vectorplasmids was determined by flow cytometry 24 hours post infection. The vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral plasmid containing the beta-globin intron.

 

Functionality of the Full Assembled Vectorplasmid Demonstrated by GOI Expression
Producing recombinant virus particles for therapeutical applications is, besides specific cell targeting, purification and quantification assays of AAV-2, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex biological system of the Adeno-associated virus serotype 2 was examined by an exhaustive literature search. Subsequently, the essential components for AAV-2 particle production were extracted and redesigned to match the iGEM standard.
The provided tripartite system is independent of a superinfection  of Adeno- or herpes simplex viruses since the genes encoding the required helper-proteins are co-transfected. Inside the eukaryotic host cell, the DNA sequence containing the inverted terminal repeats (ITRs) is extracted and later encapsidated into the preformed capsids after production of single-stranded DNA. Consequently, this plasmid is known as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs (ITRs, BBa_K404100 and BBa_K404101) and regulate transgene expression. The vector plasmid containing the desired gene of interest is cotransfected with the RepCap plasmid ( BBa_K404001, BBa_K404102 or BBa_K404103) and the pHelper plasmid. To obtain the fully assembled vector plasmid, several assembly steps have to be performed.  
After assembly of plasmids containing all required elements, vector plasmid functionality was confirmed in cell culture. AAV-293 cells stably expressing the E1A and E1B proteins were transfected with three plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72 hours post transfection and the tumor cell line HT1080 was transduced with the recombinant viral vectors encapsidating the gene of interest mVenus ( BBa_I757008).
In the beginning, the iGEM team Freiburg_Bioware 2010 used a commercial vector plasmid, pAAV-MCS (Stratagene), to determine whether virus particle production by AAV-293 cells could be achieved. iGEM RFC 25 restriction enzyme sites were introduced and the fluorescent protein mVenus was subcloned into this plasmid. Subsequently, this plasmid was used as a reference and compared to the assembled vector plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (abbreviated pSB1C3_mVenus, BBa_K404119). Fluorescence expression data obtained by flow cytometry analysis are shown in Figure 11 and Figure 12. Comparing mVenus expression of the reference plasmid and the pSB1C3_mVenus plasmid revealed that biological functionality of the reassembled plasmid was preserved.

pSB1C3_mVenus ( BBa_K404119)

pAAV_mVenus (reference)

Figure 12: Flow cytometry analysis of fluorescent protein expression in transduced HT1080 cells. For viral particle production, AAV-293 cells were transfected with the reassembled vector plasmid ( BBa_K404119) or the reference plasmid, respectively. A: Gating non transduced cells (control); subcellular debris and cellular aggreates can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non-transduced cells plotted against cells expressing mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5) C: Gating transduced cells (R2 ≙ R14) (plasmids used for transfection: pGOI: pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119), pHelper, pRC. D: Transduced cells plotted against cells expressing mVenus. R10 comprises transduced cells detected by mVenus fluorescence. E: Overlay of non-transduced (red) and transduced (green). F: Gating non-transduced cells (control). G: Non-transduced cells plotted against cells expressing mVenus (R5). H: Gating transduced cells (R14 ≙ R2) (plasmids used for transfection: pGOI: pAAV_mVenus, pHelper). I: Transduced cells plotted against cells expressing mVenus. R10 comprises transduced cells detected by mVenus fluorescence. J: Overlay of non-transduced (red) and transduced (green) cells plotted against mVenus expression. 

 

 

 

Figure 13: Quantification of mVenus fluorescence by flow cytometry analysis. The data source is identical to Figure 13. Compared to the reference vector plasmid, mVenus expression in HT1080 cells is enhanced in case of the assembled plasmid (pSB1C3_mVenus) 

Conclusion

Idea of the modular ‘Virus Construction Kit’ is to provide all required elements for producing recombinant, functional virus particles delivering encapsidated genes of interest to specific cells. First step was to modify and modularize the vectorplasmid comprising basically the cis-elements for replication (ITRs), a strong eukaryotic or tissue specific promoter (pCMV or phTERT), the gene of interest (fluorescent proteins or suicide genes) and the hGH termination signal. Each element was successfully cloned and reassembled resulting in functional vectorplasmids determined by flow cytometry and fluorescence microscopy analyses. Experiments have been performed with mVenus since measurement of fluorescent proteins can be easily performed and visualized. Considering the results, the iGEM team Freiburg_Bioware 2010 then tested the construct containing the suicide genes thymidine kinase and cytosine deaminase. Further details demonstrating efficient tumor killing, using prodrug-activating systems, see results page Arming.

References

Danckwardt, S., Hentze, M.W. & Kulozik, A.E., 2008. 3' end mRNA processing: molecular mechanisms and implications for health and disease. The EMBO journal, 27(3), 482-98. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18256699.

Dong, J.Y., Fan, P.D. & Frizzell, R.a., 1996. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Human gene therapy, 7(17), 2101-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.

Millevoi, S. et al., 2006. An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries. The EMBO journal, 25(20), 4854-64. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17024186.

Nott, A., Meislin, S.H. & Moore, M.J., 2003. A quantitative analysis of intron effects on mammalian gene expression. RNA (New York, N.Y.), 9(5), 607-17. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.

 


Move to Top

 

Overview of RepVP123 plasmid

Modularization: Overview

In our terminology the term “RepVP123” encompasses the whole AAV2 genome excluding the ITRs. The rep locus comprises four proteins related to genome replication while the cap locus codes for the proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which are required for viral capsid assembly. Source of the RepVP123 BioBrick supplied within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the wild-type AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In order to introduce iGEM standard and additionally enabling the possibility to modify the viral capsid via integration of certain motives within the viral loops 453 and 587 a total of twelve mutations within RepVP123 (see Figure 1) and additionally two mutations within the pSB1C3 backbone were performed either by Site-Directed Mutagenesis (SDM) or by ordering and cloning of specifically designed gene sequences matching the required demands. Modifying the pSB1C3 led to iGEM team Freiburg_Bioware’s variant of this backbone, pSB1C3_001.

Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\RepCap_complete_modifications_arrows.jpg

Figure 1 Mutations implemented into RepVP123 in order to establish both iGEM standard and loop insertion capability. Green arrows indicate integrated restriction sites, red red arrows indicate deleted restriction sites. KpnI was deleted first and reinstated afterwards. (see text).


 

Plasmid name:

Functionality (determinded in cell culture via transduction and flow cytometry ):

4x mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))

inserted rep fragment

inserted cap fragment

reinstated KpnI

pAAV

pSB1C3_001

HSPG-ko

pAAV_RC (wild-type)

yes

 

 

 

 

x

 

 

pAAV_RC_4x mutant

yes

x

 

 

 

x

 

 

pAAV_RC_inserts

no

x

x

x

 

x

 

 

pAAV_RC_Cap

yes

x

 

x

 

x

 

 

pAAV_RC_RepVP123

yes

x

x

x

x

x

 

 

pSB1C3_RepVP123_ p5TATAless

yes

x

x

x

x

 

x

 

pSB1C3_RepVP123_ HSPG-ko_p5TATAless

yes

x

x

x

x

 

x

x

Figure 2 Table contains complete overview about all plasmids containing RepVP123 which were used by iGEM team Freiburg_Bioware 2010.

Modularization: Removing iGEM restriction sites and establishing loop insertion capability

Modifications in Rep

Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg

Figure 3 Restriction sites within the wild-type rep gene sequence, which were removed via cloning of synthetized rep gene fragment into the plasmid. The red box indicates the region spanned by the synthetic sequence.

Making the RepVP123 wild-type compatible with the iGEM standards required the removal of five restriction sites (see Figure 1). This was achieved using site-directed mutagenesis for PstI (position 310) and PstI (4073). The remaining three iGEM restriction sites EcoRI (1578), PstI (1773) and EcoRI (1796) were replaced by a synthetic gene fragment, since the rep ORF contained these restriction sites in close proximity to each other plus an additional KpnI restriction site which was also not desired (see Figure 2). This gene fragment was cloned into the rep gene using HindIII and SwaI, which are single-cutting restriction enzymes adjacent to the target area. Additionally, BamHI (859) and SalI (1239) were removed, because these enzymes were required for genetically inserting the loop modifications in VP123.

Modifications in VP123

In order to implement the restriction sites necessary for targeting via loop insertions, the gene coding for the VP proteins was modified as well. The introduction of these restriction required up to four base mutations in a row, hence it was decided to synthesize this gene fragment and replace the wild-type sequence in RepVP123 as well.

Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg

Figure 4 Restriction sites within cap sequence showing introduced loop insertion restriction sites into cap to enable cloning of targeting or purification motifs into both 453 and 587 loops. Again, the red box indicates gene sequence which was synthetized.

 

Figure 5 Results for transduction efficiency measured by flow cytometry. Fluorescence is measured in surviving cells. The tested RepVP123 containing four point mutation to delete iGEM and loop insertion restriction sites does not show any difference within mVenus expression compared to the wild-type and therefore can be verified working.

Alongside to creating an iGEM compatible plasmid, infectivity of the modified construct was tested in cell culture via flow cytometry. Then experiments confirmed that single cloning steps did not interfere with natural viral infectivity at first (see above). But cloning of the synthesized rep gene fragment into the plasmid dramatically reduced transduction efficiency as detected by flow cytometry. Scrutinizing each mutation and its potential impact, suggested that abolished transduction was related to the mutation, which removed the KpnI (1721) site. This site is located within a splice site, which is crucial for the Rep proteins, and thus even silent mutations may interfere with virus production (see extra topic “Rep proteins”).


 xxx


 


Description: Fluorescence of HT1080 cells transduced with FRAGE AN ADRIAN:WELCHES GOI? and P325 and P326 both containing RepCap in which the synthetized gene sequences were inserted. As control,pAAV_RC (P50) containing wtRepCap was used. mVenus expression within cells clearly state that tested constructs does not meet expectations.

Figure 8 AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus _hGH_[AAV2]-right-ITR and pAAV_RC_inserts (see above) or pAAV_RC (see below) providing essential genes and proteins for producing viral particles. After 48 hour post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytomery 24 hours post infection. Fluorescence is measured in surviving cells. Results show that insertion of both rep and cap syntheses disrupts viral infectivity.

Therefore, additional constructs were designed containing only the synthetic cap sequence or both, the synthesized sequences plus a re-mutation of KpnI (1721), in order to re-establish the wild-type splice site within the rep ORF. Results from cell culture obtained via FACS revealed that in fact the poor results were related to the KpnI restriction site deletion. Both constructs showed a transduction efficiency corresponding to the unmodified wild-type RepVP123’s transduction efficiency.

Figure 9 Fluorescence of cells transduced with mVenus carrying rAAV measured by flow cytometry. AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR and RepVP123 constructs providing essential genes and proteins for producing viral particles. 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytometry 24 hours post infection. Results show that cap integration does not influence infectivity. Fluorescence is measured in surviving cells. Recreation of KpnI within rep splice site recovers transduction efficiency.

 

Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001

To fulfill iGEM requirements all plasmids need to be submitted in pSB1C3, therefore primers were ordered for amplifying RepVP123 containing all modifications done so far by PCR and cloning the into pSB1C3. Still, pSB1C3 contains two restriction sites for SspI and PvuII restriction enzymes in its CAT marker. Since these are necessary for cloning ViralBricks in this vector, the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM Headquarters to implement a new standard for the pSB1C3 backbone which was named pSB1C3_001. Both restriction sites interfering with ViralBrick insertions were mutated to make SspI and PvuII single-cutters (see method development).

Figure 10 Comparison of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and PvuII are marked by red boxes.

RepVP123 containing both rep and cap synthetic gene fragments including the re-mutation of KpnI and the downstream p5TATA-less promotor was cloned into the newly constructed pSB1C3_001. Testing this newly assembled plasmid in cell culture revealed unexpected data. Not only did the newly assembled plasmid work (see Figure 10), but in comparison to pAAV containing the same RepVP123 construct, pSB1C3_001 showed an about 3 times higher transduction efficiency. Although exact reasons are still unknown, these results are probably related to the reduced length of pSB1C3_001 compared to the original pAAV plasmid of approximately 1000 base pairs.

 

Figure 11 AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR providing essential genes and proteins for producing viral particles. After 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytomery after 24 hours post infection. Fluorescence is measured in surviving cells. Results showed functionality of RepVP123 within pSB1C3_001 vector and additionally increased transduction efficiency.

Turning-off natural tropism: HSPG-knock-out

Shutting-down the natural viral tropism is essential for targeting specifically tumor cells and not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware 2010 decided to knock-out the viral natural tropism delivered by the heperan sulfate proteoglycan-(HSPG) binding site within the viruses 587 loop. The knock-out was cloned by designing primers containing the required base exchanges and performing a SDM. Like performed before, this RepVP123 variant was tested in cell culture as well and evaluated by flow cytometry. Results show that mutation of HSPG-binding motif has severe impact on transduction efficiency thus enabling a viral particle carrying this knock-out and additional targeting motifs, e.g. within the loops or presented via N-terminal fusion to bind target cells’ receptors and therefore infecting target cells at a much higher rate compared to unspecific infection of other cell types within an organism (see Figure 12).

To quantify differences in infectivity, the infectious titer of viral particles built-up of RepVP123 with and without HSPG binding motif was determined by qPCR (see Figure 14) for different cell lines. Results show that the implemented HSPG-knock-out verifies results obtained from flow cytometry, infectious titers severely compared to RepVP123 with intact HSPG binding motif.

Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\HSPG-ko_modified.jpg

Figure 12 Alignment of 587 loop within viral VP123: The upper sequence shows a strand containing the HSPG binding motif (AGA, in red boxes), the lower sequence contains the HSPG-ko introduced by the iGEM team Freiburg_Bioware 2010 (GCT and GCC, blue boxes).

 

Figure 13 Transduction efficiency of HT1080 cells measured by flow cytometry. Fluorescence is measured in surviving cells. Knock-out of HSPG binding motif greatly reduces transduction efficiency compared to RepVP123 containing the motiv.

 

Figure 14 Infectious titers of RepVP123 with and without natural HSPG binding motif tested in different cell lines via qPCR. Shutting-down the HSPG binding motif reduces infectious titer in both HT1080 and HeLa cell lines. For A431 cells, no infectious titer could be detected via qPCR, which is probably related to poor transduction efficiency of A431 cells.

Move to Top