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2010-11-21T16:22:20Z
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Hanna
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Hanna at 14:01, 27 October 2010
2010-10-27T14:01:34Z
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Hanna: New page: {{:Team:Freiburg_Bioware/Head}} {{:Team:Freiburg_Bioware/jquery}} {{:Team:Freiburg_Bioware/menu_home}} <html> <p class="MsoToc2"><span lang="DE"><a href="#_Toc275953546"><span lang="EN-U...
2010-10-27T13:48:53Z
<p>New page: {{:Team:Freiburg_Bioware/Head}} {{:Team:Freiburg_Bioware/jquery}} {{:Team:Freiburg_Bioware/menu_home}} <html> <p class="MsoToc2"><span lang="DE"><a href="#_Toc275953546"><span lang="EN-U...</p>
<p><b>New page</b></p><div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<br />
<html><br />
<p class="MsoToc2"><span lang="DE"><a<br />
href="#_Toc275953546"><span lang="EN-US">Highlighting<br />
N-terminal Fusion:</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953547"><span lang="EN-US">Design</span></a></span></p><br />
<p class="MsoToc4"><span lang="DE"><a<br />
href="#_Toc275953548"><span lang="EN-US">VP2<br />
Fusion</span></a></span></p><br />
<p class="MsoToc4"><span lang="DE"><a<br />
href="#_Toc275953549"><span lang="EN-US">VP1<br />
Insertion</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953550"><span lang="EN-US">Cloning<br />
Verified by Colony PCR</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953551"><span lang="EN-US">Fluorescence<br />
Analysis of Viral Stocks</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953552"><span lang="EN-US">Concentrating<br />
VP1UP_NLS_mVenus_VP2/3 Viruses</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953553"><span lang="EN-US">Nuclear<br />
Localization by Fluorescence Microscopy</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953554"><span lang="EN-US">Transduction<br />
Efficacy by Flow Cytometry</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953555"><span lang="EN-US">Infectious<br />
Titer by qPCR</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953556"><span lang="EN-US">Killing<br />
cells: Time-Lapse</span></a></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h2><a name="_Toc275953546">Highlighting N-terminal<br />
Fusion:</a></h2><br />
<h3><a name="_Toc275953547">Design</a></h3><br />
<h4><a name="_Toc275953548">VP2 Fusion</a></h4><br />
<p class="MsoNormal">The Freiburg2010 team fused motifs<br />
which are desired to be<br />
surface exposed to the N-terminus of VP2/3 open reading frame, avoiding<br />
steric<br />
hindrance by connecting them with linker. We ensured in frame coupling<br />
by<br />
designing or using the referring parts in the Freiburg RFC25 standard,<br />
thus<br />
creating fusion constructs via NgoMIV and AgeI. For retargeting AAV2<br />
virus<br />
particles towards EGFR over expressing cancer cells we investigated the<br />
Z<sub>EGFR:1907</sub><br />
Affibody and the E01 DARPin – in combination with VP2/3_587-KO (HSPG<br />
knock<br />
down) – as surface exposed motifs. We also created so called<br />
“all-in-one” virus<br />
particles by fusing VP2/3_His-Tag or VP2/3_BAP via Middle Linker to the<br />
Affibody. Purification or imaging approaches could be conducted by<br />
His-Tag or<br />
CFP motifs, respectively. All constructs were cloned downstream of the<br />
CMV<br />
promoter (Fig.1).</p><br />
<p class="MsoNormal"><b>Table 1: List of VP2 fusion<br />
parts</b></p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/b/bf/Freiburg10_SchemaVP2Fusion.PNG"<br />
src="https://static.igem.org/mediawiki/2010/b/bf/Freiburg10_SchemaVP2Fusion.PNG"></p><br />
<p class="MsoNormal"></p><br />
<br><br />
<p class="MsoNormal">&nbsp;&nbsp; <img<br />
alt="https://static.igem.org/mediawiki/2010/2/25/Freiburg10_Overview_VP2Fusion.png"<br />
src="https://static.igem.org/mediawiki/2010/2/25/Freiburg10_Overview_VP2Fusion.png"></p><br />
<p class="MsoNormal"><b>Figure 1: Cloning scheme of<br />
VP2 fusion</b></p><br />
<p class="MsoNormal">First of all we fused the Affibody<br />
ZEGFR:1907 to the Short-,<br />
Middle-, Long- or SEG Linker and cloned the resulting constructs<br />
downstream of the<br />
CMV promoter. Afterwards either unmodified VP2/3 or HSPG affinity knock<br />
down<br />
VP2/3 were fused to the different linkers. Further on CFP and His-Tag<br />
were<br />
coupled to VP2/3 by the Middle Linker (Fig. 2).</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/6/6a/Freiburg10_GelVP2Fusion2tog.PNG"<br />
src="https://static.igem.org/mediawiki/2010/6/6a/Freiburg10_GelVP2Fusion2tog.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><span<br />
style=""><span style="">&nbsp; </span></span><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
2:</span></b><span style="font-size: 10pt;"><br />
Agarose Gel Electrophoresis.<br />
Fusion of VP2/3 to CMV_Affibody_Linker or CMV_CFP_MiddleLinker<br />
constructs.</span></p><br />
<p class="MsoNormal">We conducted three fragment ligations<br />
with the DARPin E01,<br />
MiddleLinker_VP2/3 or MiddleLinker_VP2/3(587KO) and pSB1C3_CMV plasmid<br />
(Fig.<br />
3).</p><br />
<p class="MsoNormal">&nbsp;<img<br />
alt="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_GelVP2Fusion2tog2.PNG"<br />
src="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_GelVP2Fusion2tog2.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
3:</span></b><span style="font-size: 10pt;"><br />
Agarose Gel Electrophoresis. <b style="">A)</b><br />
Three fragment ligation of DARPin<br />
fused to N-terminus of VP2/3. <b style="">B)</b><br />
Test digestion verified successful cloning.<o:p></o:p></span></p><br />
<p class="MsoNormal">For 100 % replacement of wildtype VP2<br />
the fusion plasmid was<br />
co-transfected to the Rep/Cap plasmid which contained a start codon<br />
mutation of<br />
VP2. Wildtype VP2 knock-out was achieved by site-directed mutagenesis<br />
(Fig. 4).<br />
</p><br />
<p class="MsoNormal"><span<br />
style="position: absolute; z-index: 251659264; left: 0px; margin-left: 295px; margin-top: 26px; width: 42px; height: 99px;"></span></p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-out;"<br />
alt="https://static.igem.org/mediawiki/2010/e/ec/Freiburg10_VP2KO.PNG"<br />
src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg10_VP2KO.PNG"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 4:<br />
Knock out of VP2</span></b> in the Cap coding region of<br />
Rep/Cap plasmid:<br />
Knock-out of start codon via site-directed mutagenesis (note the C in<br />
the sequence).</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h4><a name="_Toc275953549">VP1 Insertion</a></h4><br />
<p class="MsoNormal">We inserted three diffent motifs into<br />
the VP1 open reading<br />
frame. They were fused, together with the unique uspstream region of<br />
VP1<br />
(VP1up) and a nuclear localization sequence (NLS), according to the<br />
RFC25<br />
standard to VP2/3 (Fig. 2). Again, all of these parts were driven by<br />
CMV<br />
promoter.</p><br />
<p class="MsoNormal"><b>Table 2: List of VP1<br />
insertion parts</b></p><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/1/1b/Freiburg10_VP1InsertionSchema.PNG"<br />
src="https://static.igem.org/mediawiki/2010/1/1b/Freiburg10_VP1InsertionSchema.PNG"><br />
<p class="MsoNormal"></p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/6/64/Freiburg10_Overview_VP1Insertion.png"<br />
src="https://static.igem.org/mediawiki/2010/6/64/Freiburg10_Overview_VP1Insertion.png"<br />
height="656" width="875"></p><br />
<p style="font-weight: bold;" class="MsoNormal">Figure<br />
5: Cloning scheme of VP1<br />
insertion</p><br />
<p class="MsoNormal">First VP1up-NLS was fused to the<br />
surface exposed motif –<br />
Affibody, His-Tag or mVenus – and the resulting constructs were cloned<br />
downstream of the CMV promoter. This was followed by either fusing<br />
unmodified<br />
VP2/3 or HSPG affinity knock down VP2/3 (Fig. 6) to the different kinds<br />
of<br />
motifs.</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Gel_VP1ins.png"<br />
src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Gel_VP1ins.png"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
6:<br />
Agarose Gel Electrophoresis.</span></b><span<br />
style="font-size: 10pt;"> Fusion of VP2/3(587KO) to<br />
pCMV_VP1up_NLS_ZEGFR:1907,<br />
pCMV_VP1up_NLS_6xHis or pCMV_VP1up_NLS_mVenus<o:p></o:p></span></p><br />
<p class="MsoNormal">We designed site-directed mutagenesis<br />
primer for the VP1<br />
start codon modification in the Rep/Cap plasmid in order to ensure 100<br />
%<br />
replacement with modified VP1 proteins (Fig. 7).</p><br />
<p class="MsoNormal">&nbsp;<img<br />
style="cursor: -moz-zoom-out;"<br />
alt="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_VP1KO.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_VP1KO.PNG"></p><br />
<p class="MsoNormal"><span<br />
style="position: absolute; z-index: 251660288; left: 0px; margin-left: 252px; margin-top: 5px; width: 42px; height: 99px;"></span></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 7:<br />
Knock out of VP1</span></b> in Cap coding region of Rep/Cap<br />
plasmid: Knock-out<br />
of start codon via site-directed mutagenesis.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953550">Cloning Verified by<br />
Colony PCR</a></h3><br />
<p class="MsoNormal">Big amounts of samples necessitated<br />
fast and exact analysis<br />
of cloning. For this purpose colony PCR was conducted. We verified<br />
successful<br />
VP2/3 fusion by using primer that exclusively bound to a specific<br />
location in<br />
the VP2/3 sequence (Fig. 8). In the case of successful ligation a 880<br />
bp<br />
fragment was amplified via Taq polymerase.</p><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/b/b4/Freiburg10_Primer_ColonyPCR.png"<br />
src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg10_Primer_ColonyPCR.png"><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 8:</span></b><br />
Primer Cap 3500 for and VP 4200 rev. Colony PCR using these primer<br />
results in a<br />
880 bp fragment</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Figure 9 demonstrates a colony PCR<br />
example: VP2 fusion<br />
constructs containing VP2/3(587) sequence and the so called “All in<br />
One”<br />
constructs were cloned into pSB1C3_CMV. Successful cloning could be<br />
verified<br />
(positive control: pAAV_RC; negative control: pSB1C3_lITR).</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_ColonyPCR.png"<br />
src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_ColonyPCR.png"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
9: Colony<br />
PCR</span></b><span style="font-size: 10pt;"><br />
of VP2<br />
N-terminal fusion (Affibody_Linker, CFP_MiddleLinker,<br />
His-Tag_MiddleLinker:<br />
samples 1 - 6) and “All-in-One” constructs (samples 7 – 10).<o:p></o:p></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953551">Fluorescence Analysis of<br />
Viral Stocks</a></h3><br />
<p class="MsoNormal">We wanted to develop a protocol for<br />
harvesting as much<br />
viruses as possible. For this purpose we took advantage of viruses<br />
containing<br />
mVenus fused to VP1. In order to find out whether most viruses were<br />
located in<br />
the cell pellet, cell suspensions were centrifuged at 10.000 g or<br />
20.000 g.<br />
Afterwards mVenus fluorescence was measured at 526 nm. The resulting<br />
values were<br />
compared to cell culture supernatant, 20.000 g centrifuged supernatant<br />
and<br />
resuspended cell pellet (Fig. 10).</p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_test_supernatant_pellet.png"<br />
src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_test_supernatant_pellet.png"<br />
height="656" width="875"> </p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
10: Fluorescence signal of mVenus at 526 nm</span></b>,<br />
inserted into the virus<br />
capsid. Transduced cells were resuspended, pelleted or supernatant was<br />
taken in<br />
order to determine the location of most viruses. Control: DMEM.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953552">Concentrating<br />
VP1UP_NLS_mVenus_VP2/3 Viruses</a></h3><br />
<p class="MsoNormal">For further analysis, viral stocks<br />
originating from cell<br />
culture supernatant needed to be concentrated. Therefore<br />
ultrafiltration with<br />
protein concentrators (Sartorius VivaSpin 20; 20.000Da MWCO) was<br />
conducted.<br />
Figure 11 shows fluorescence microscopy image of an aliquot from<br />
ultrafiltrated<br />
cell culture supernatant / cell lysate containing<br />
VP1up_NLS_mVenus_VP2/3<br />
viruses.</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/d/d9/Freiburg10_mVenusDrop.PNG"<br />
src="https://static.igem.org/mediawiki/2010/d/d9/Freiburg10_mVenusDrop.PNG"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
11: Fluorescence Microscopy</span></b>. A) Brightfield<br />
picture of an aliquot of<br />
concentrated VP1up_NLS_mVenus_VP2/3 containing virus stock. B) At 505<br />
nm<br />
excitation mVenus fluorescence of the virus particles could be<br />
detected. C)<br />
Merged image</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953553">Nuclear Localization by<br />
Fluorescence Microscopy</a></h3><br />
<p class="MsoNormal">AAV-293 cells were transfected with a<br />
50:50 ratio of the<br />
Rep/Cap(VP1KO) to the CMV_VP1_NLS_mVenus_VP2/3 plasmid. We packaged<br />
mCherry,<br />
driven by the CMV promoter, into the virus capsids and followed protein<br />
expression<br />
via fluorescence microscopy. 30 hours post transfection mCherry<br />
fluorescence<br />
was detectable in the whole cytosol of the successfully transfected<br />
cells,<br />
demonstrating that DNA located between the AAV2 ITRs is already<br />
transcribed in<br />
the producer cell line. In contrast to that mVenus fluorescence signal<br />
could be<br />
observed only in the nuclei (Fig. 12). This indicated that the nuclear<br />
localization sequence targets the single VP1_NLS_mVenus_VP2/3 proteins<br />
efficiently to the cell nucleus, where assembly and packaging of the<br />
virus<br />
particles takes place.&nbsp; </p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_NLS_mVenus_mCherry.png"<br />
src="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_NLS_mVenus_mCherry.png"<br />
height="656" width="875"></p><br />
<p class="MsoNormal" style="line-height: normal;">&nbsp;<br><br />
<b style=""><span style="font-size: 10pt;">Figure<br />
12:<br />
Fluorescence Microscopy. A) </span></b><span<br />
style="font-size: 10pt;">Brightfiled picture. <b style="">B)</b><br />
Excitation at 555 nm showed mCherry signal in the cytosol. <b<br />
style="">C)</b> Excitation at 505 nm revealed mVenus<br />
fluorescence in the nuclei, indicating functionality of the nuclear<br />
localization sequence inserted – together with mVenus – into VP1. <b<br />
style="">D)</b> Merged image.</span></p><br />
<p class="MsoNormal" style="line-height: normal;"><span<br />
style="font-size: 10pt;"><o:p></o:p></span></p><br />
<h3><a name="_Toc275953554">Transduction Efficacy by<br />
Flow Cytometry</a></h3><br />
<p class="MsoNormal">For determination of transduction<br />
efficacy flow cytometry<br />
analysis was conducted. The Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
was fused with SEG-, middle- and long<br />
linker to the VP2/3 open reading frame (ORF) in order to investigate<br />
differences in infection efficacy due to different linker lengths.<br />
250.000<br />
AAV-293 cells were transfected with 1 µg total DNA. Different ratios of<br />
VP2<br />
fusion constructs in respect to the Rep/Cap plasmid were<br />
co-transfected. 72<br />
hours post transfection viruses were harvested and two different cell<br />
lines,<br />
HT1080 and A431, were transduced with 1 mL virus stock. By<br />
encapsulating mVenus<br />
coding sequence, the amount of transduced cells could be determined via<br />
flow<br />
cytometry.</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_FACS1.PNG"<br />
src="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_FACS1.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 13: Flow<br />
cytometry.</span></b><span style=""> Test of<br />
transduction efficiency with HT1080<br />
and A431 cells by detecting mVenus expression from <b style="">Z<sub>EGFR:1907</sub>_<span<br />
style="">Middlelinker</span>_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non-transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span></p><br />
<p class="MsoNormal"> <img<br />
alt="https://static.igem.org/mediawiki/2010/9/9f/Freiburg10_FACS2.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/9f/Freiburg10_FACS2.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 14: Flow<br />
cytometry.</span></b><span style=""><br />
Investigating transduction efficiency with<br />
HT1080 and A431 cells by detecting mVenus expression from <b>Z<sub>EGFR:1907</sub>_SEG_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non-transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span></p><br />
<p class="MsoNormal" style="line-height: normal;"><img<br />
alt="https://static.igem.org/mediawiki/2010/7/77/Freiburg10_FACS3.PNG"<br />
src="https://static.igem.org/mediawiki/2010/7/77/Freiburg10_FACS3.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 15: Flow<br />
cytometry.</span></b><span style=""><br />
Investigating transduction efficiency with HT1080<br />
and A431 cells by detecting mVenus expression from <b style="">Z<sub>EGFR:1907</sub>_<span<br />
style="">LongLinker</span>_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span><b><span style="font-size: 12pt;"><o:p></o:p></span></b></p><br />
<span style=""><o:p></o:p></span><br />
<p class="MsoNormal">Additionally viruses containing a<br />
His-Tag or CFP, fused via<br />
Middle Linker to the VP2/3 ORF, were analyzed. Figure 16 overviews<br />
transduction<br />
efficacy of all constructs, transfected in a 10:90, 25:75 and 50:50<br />
ratio in<br />
respect to Rep/Cap plasmid.&nbsp;&nbsp; </p><br />
<p class="MsoNormal">&nbsp;<img<br />
alt="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png"<br />
src="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png"></p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/1/15/Freiburg10_FACS_VP2_A431.png"<br />
src="https://static.igem.org/mediawiki/2010/1/15/Freiburg10_FACS_VP2_A431.png"><br><br />
<b>Figure 16: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive HT1080 and A431 cells, infected with virus<br />
particles<br />
consisting of different ratios of VP2 fusion constructs in respect to<br />
Rep/Cap<br />
plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Transduction of HT1080 cells revealed<br />
that all viral<br />
particles regardless of which motifs inserted into the capsids remained<br />
infectious with efficacies up to 85 %. In general the amount of mVenus<br />
positive<br />
cells decreased only slightly when harboring more modified VP2<br />
subunits. This<br />
indicates that larger peptides could be inserted into the AAV2 capsids<br />
without<br />
affecting virus assembly and packaging. A431 cells, which overexpress<br />
EGF<br />
receptor, were generally transduced with reduced efficacy. </p><br />
<p class="MsoNormal">Further on the Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
or mVenus were inserted into the VP1<br />
ORF together with a nuclear localization signal. 250.000 AAV-293 cells<br />
were<br />
transfected with 1 µg total DNA and different ratios of VP1 insertion<br />
constructs in respect to the Rep/Cap(VP1KO) plasmid were<br />
co-transfected. 72<br />
hours post transfection viruses were harvested and HT1080 and A431<br />
cells were<br />
transduced. 48 hours later the number of transduced, or to be precise<br />
mVenus<br />
positive, cells was determined via flow cytometry (Fig. 17 a, b).</p><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_FACS_VP1Ins_HT1080.png"<br />
src="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_FACS_VP1Ins_HT1080.png"><br />
<p class="MsoNormal"><b>Figure 17 a: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive HT1080 cells, infected with virus particles<br />
containing different ratios of VP1 insertion constructs in respect to<br />
Rep/Cap(VP1KO) plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/5/5b/Freiburg10_FACS_VP1Ins_A431.png"<br />
src="https://static.igem.org/mediawiki/2010/5/5b/Freiburg10_FACS_VP1Ins_A431.png"></p><br />
<p class="MsoNormal"><b>Figure 17 b: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive A431 cells, infected with virus particles<br />
containing<br />
different ratios of VP1 insertion constructs in respect to<br />
Rep/Cap(VP1KO)<br />
plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Again results revealed that all virus<br />
particles remained<br />
infectious, regardless of whether inserting the Affibody or mVenus.<br />
This<br />
indicated that VP1 tolerated larger peptides inserted downstream of its<br />
unique<br />
N-terminal region and that this modification still allowed virus<br />
assembly and<br />
packaging.</p><br />
<h3><a name="_Toc275953555">Infectious Titer by qPCR</a><br />
</h3><br />
<p class="MsoNormal">We transfected 250.000 AAV-293 cells<br />
with 1 µg of total DNA<br />
composed of equal amounts of Rep/Cap, pHelper and vector plasmid. VP2<br />
fusion or<br />
VP1 insertion plasmids were co-transfected with two different ratios in<br />
respect<br />
to Rep/Cap(VP1KO) or Rep/Cap(VP2KO):&nbsp; 25&nbsp;% VP2 fusion<br />
or VP1 insertion<br />
proteins allow better assembly and packaging of the virus particles,<br />
compared<br />
to 50 % VP2 fusion or VP1 insertion proteins which increase the chance<br />
of&nbsp;<br />
integration into the capsids. </p><br />
<p class="MsoNormal">We created viruses containing the Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
Affibody fused<br />
to VP2 or inserted into VP1 and the DARPin E01 fused to VP2. These<br />
types of<br />
AAV2 particles were produced in two versions: With or without HSPG<br />
binding<br />
affinity knock down (587KO). </p><br />
<p class="MsoNormal">Viruses were harvested three days<br />
post transfection. The genomic<br />
titer was determined via qPCR by amplification of a specific sequence<br />
located<br />
in the CMV promoter of the vector plasmid (Table 3).</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Table 3:<br />
Quantitative Real-Time PCR.</span></b> Determination of<br />
genomic titer. Data<br />
were corrected for negative control value.</p><br />
<div align="center"><br />
<table class="MsoNormalTable"<br />
style="border: medium none ; width: 450.2pt; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"<br />
width="600"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Co-transfected<br />
Construct</span></b></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Ratio</span></b></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Genomic<br />
Titer /1ml</span></b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Corrected<br />
For Negative Control</span></b></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">2,20E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">2,39E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,17E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,44E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">CFP_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,63E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">CFP_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,67E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6xHis_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,37E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6xHis_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,38E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,52E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,50E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6,98E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,30E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_6xHis_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,25E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_6xHis_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,65E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">4,36E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,93E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,00E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,99E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Control: Rep/Cap</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">100%</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,55E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Control: Rep/Cap(587KO)</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">100%</p><br />
</td><br />
<td<br />
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: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,39E+08</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">We investigated transduction of<br />
different cell lines. For<br />
this purpose 100.000 HT1080, HeLa or A431 cells were seeded and<br />
transduced with<br />
50 µL virus stock and harvested 24 hours later.&nbsp; Infectious<br />
titers were<br />
determined via qPCR and normalized to the genomic titers (Fig. 17-19).</p><br />
<p class="MsoNormal">Figure 18 shows infection efficacy of<br />
DARPin exposing<br />
viruses. Transduction of HT1080 cells was almost not affected as long<br />
as<br />
binding to HSPG was not knocked down. HeLa cells were also infected<br />
less<br />
efficient compared to the controls. However, A431 cells which<br />
overexpress EGFR<br />
were not infected by the controls. Transduction is rescued by<br />
integration of<br />
the DARPin into the virus capsid. By additionally knocking down the<br />
HSPG<br />
binding affinity these cells are transduced 10 times better, reaching<br />
wild type<br />
capsid HT1080 infection efficacy. These results indicated that specific<br />
re-targeting of AAV2 virus particles towards EGFR over expressing tumor<br />
cells<br />
was achieved by N-terminal fusion of targeting motifs to VP2.<br><br />
<br><br />
<br><br />
<img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_qPCR_DARPin.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_qPCR_DARPin.PNG"<br />
height="656" width="875"><br />
</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Figure 18:<br />
DARPin E01 VP2 Fusion.</span></b> Infectious titers were<br />
determined with or<br />
without HSPG knock down for HT1080, HeLa and A431 cells.<br />
Control:Rep/Cap<br />
plasmid with and without HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">We additionally obtained similar<br />
results for Affibody<br />
exposing viruses: A431 cell transduction could also be rescued by<br />
capsid<br />
integration of the Affibody. Again infection efficacy was increased by<br />
knocking<br />
down the natural tropism of the AAV2 viruses towards HSPG (Fig. 19).<br />
These data<br />
emphasized the functionality of the VP2 fusion constructs for<br />
specifically<br />
targeting tumor cells for therapeutic or imaging<br />
applications.&nbsp; </p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_qPCR_VP2Fus.PNG"<br />
src="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_qPCR_VP2Fus.PNG"<br />
height="656" width="875"><br><br />
<b><span style="font-size: 10pt; line-height: 200%;">Figure<br />
19:<br />
Affibody Z<sub>EGFR:1907</sub> VP2 Fusion.</span></b><br />
Infectious titers were<br />
determined with or without HSPG knock down for HT1080, HeLa and A431<br />
cells.<br />
Control:Rep/Cap plasmid with and without HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Figure 20 repesents the infectivity<br />
of viruses containing<br />
VP1 inserted Affibody molecules in respect to three different cell<br />
types<br />
(HT1080, HeLa, A431). The data clearly shows that virus capsids<br />
assembled and<br />
remained infectious. These AAV2 particles also featured specific<br />
binding<br />
properties to A431 cells, validating the functionality of&nbsp; the<br />
VP1 insertion<br />
strategy. The two outlier (HeLa VP1up_NLS_Affibody_VP2/3 25:75<br />
&amp; HT1080<br />
VP1up_NLS_Affibody_VP2/3(587KO) 50:50) should be left unconsidered. </p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_qPCR_VP1Insnew.PNG"<br />
src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_qPCR_VP1Insnew.PNG"<br />
height="656" width="875"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Figure 20:<br />
Affibody Z<sub>EGFR:1907</sub> VP1 Insertion.</span></b><br />
Infectious titers were<br />
determined for 25:75 and 50:50 transfection ratios with or without HSPG<br />
knock<br />
down for HT1080, HeLa and A431 cells. Control:Rep/Cap plasmid with and<br />
without<br />
HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953556">Killing cells: Time-Lapse</a></h3><br />
<p class="MsoNormal">HT1080 and A431 cells were transduced<br />
with so called<br />
“All-in-One” viruses containing the Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
fused to VP2/3(587KO_His-Tag) and<br />
packaged with the guanylate kinase fused to the thymidine kinase coding<br />
sequence (GMK-TK). A time series of pictures was started directly after<br />
adding<br />
20 µM Ganciclovir (Fig.21).</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_TimeLapseHT1080.png"<br />
src="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_TimeLapseHT1080.png"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 21<br />
a: Time-lapse</span></b>. HT1080 (control) cells transduced<br />
with GMK-TK<br />
packaged viruses and treated with 20 µM Ganciclovir A) 0 hours, B) 7<br />
hours, C)<br />
15 hours and D) 23 hours post transduction.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/3/3e/Freiburg10_TimeLapseA431.png"<br />
src="https://static.igem.org/mediawiki/2010/3/3e/Freiburg10_TimeLapseA431.png"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 21<br />
b: Time-lapse</span></b>. A431 cells transduced with GMK-TK<br />
packaged viruses<br />
and treated with 20 µM Ganciclovir A) 0 hours, B) 7 hours, C) 15 hours<br />
and D)<br />
23 hours post transduction.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Virus and Ganciclovir treatment only<br />
slightly affect the<br />
morphology of HT1080 cells. After 23&nbsp;hours of incubation in<br />
20µM<br />
Ganciclovir the cells had almost the same appearance as at time point<br />
zero<br />
(Fig. 21 a). In comparison to that A431 epidermoid carcinoma cells were<br />
efficiently killed after transduction and Ganciclovir add-on: After 23<br />
hours<br />
nearly all cells were lysed (Fig. 21 b). These results clearly<br />
demonstrate that<br />
we were able to specifically target EGFR over-expressing tumor cells<br />
via capsid<br />
integrated Affibody and that these transduced cells were efficiently<br />
killed by<br />
expressing the GMK-TK which converted prodrug Ganciclovir into its<br />
cell-toxic monophosphate.<br />
</p><br />
</html></div>
Hanna