Team:Freiburg Bioware/Project/Results/Arming

From 2010.igem.org

(Difference between revisions)
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<div class="WordSection1">
 
<p class="MsoTocHeading">Contents</p>
<p class="MsoTocHeading">Contents</p>
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<p class="MsoToc2"><span class="MsoHyperlink"><a
+
 
-
href="#_Toc275981841"><span lang="EN-US">Arming:
+
<p class="MsoToc2"><a href="#_Toc275981841"><span lang="EN-US">Arming: Suicide
-
Suicide Genes as GOIs</span><span
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Genes as GOIs</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">1</span></a></p>
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style="color: windowtext; display: none; text-decoration: none;">.
+
 
-
</span><span
+
<p class="MsoToc3"><a href="#_Toc275981842"><span lang="EN-US">Introduction</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">1</span></a></p>
-
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
+
 
-
<p class="MsoToc3"><span class="MsoHyperlink"><a
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<p class="MsoToc3"><a href="#_Toc275981843"><span lang="EN-US">Successful Assembly
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href="#_Toc275981842"><span lang="EN-US">Introduction</span><span
+
of Vector Plasmids Carrying Suicide Genes via Cloning</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">1</span></a></p>
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style="color: windowtext; display: none; text-decoration: none;">.
+
 
-
</span><span
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<p class="MsoToc3"><a href="#_Toc275981844"><span lang="EN-US">Monitoring Efficient
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style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
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Tumor Killing by Phase-Contrast Microscopy</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">4</span></a></p>
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<p class="MsoToc3"><span class="MsoHyperlink"><a
+
 
-
href="#_Toc275981843"><span lang="EN-US">Successful
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<p class="MsoToc3"><a href="#_Toc275981845"><span lang="EN-US">Quantitative
-
Assembly of Vector Plasmids Carrying Suicide Genes via
+
Analysis of Cell Death by Flow Cytometry</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">6</span></a></p>
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Cloning</span><span
+
 
-
style="color: windowtext; display: none; text-decoration: none;">.
+
<p class="MsoToc3"><a href="#_Toc275981846"><span lang="EN-US">Titrating
-
</span><span
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Ganciclovir Concentrations for Efficient Cell Killing by Cytotoxicity Assays</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">8</span></a></p>
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style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
+
 
-
<p class="MsoToc3"><span class="MsoHyperlink"><a
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<p class="MsoToc3"><a href="#_Toc275981847"><span lang="EN-US">Killing Untransduced
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href="#_Toc275981844"><span lang="EN-US">Monitoring
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Tumor Cells via Bystander Effect</span><span style="color: windowtext; display: none; text-decoration: none;"> </span><span style="color: windowtext; display: none; text-decoration: none;">13</span></a></p>
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Efficient Tumor Killing by Phase-Contrast Microscopy</span><span
+
 
-
style="color: windowtext; display: none; text-decoration: none;">.
+
<p class="MsoToc3"><a href="#_Toc275981848"><span lang="EN-US">Conclusions</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">16</span></a></p>
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</span><span
+
 
-
style="color: windowtext; display: none; text-decoration: none;">4</span></a></span></p>
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<p class="MsoToc3"><a href="#_Toc275981849"><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;">16</span></a></p>
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<p class="MsoToc3"><span class="MsoHyperlink"><a
+
 
-
href="#_Toc275981845"><span lang="EN-US">Quantitative
+
-
Analysis of Cell Death by Flow Cytometry</span><span
+
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style="color: windowtext; display: none; text-decoration: none;">.
+
-
</span><span
+
-
style="color: windowtext; display: none; text-decoration: none;">6</span></a></span></p>
+
-
<p class="MsoToc3"><span class="MsoHyperlink"><a
+
-
href="#_Toc275981846"><span lang="EN-US">Titrating
+
-
Ganciclovir Concentrations for Efficient Cell Killing by
+
-
Cytotoxicity Assays</span><span
+
-
style="color: windowtext; display: none; text-decoration: none;">.
+
-
</span><span
+
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style="color: windowtext; display: none; text-decoration: none;">8</span></a></span></p>
+
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<p class="MsoToc3"><span class="MsoHyperlink"><a
+
-
href="#_Toc275981847"><span lang="EN-US">Killing
+
-
Untransduced Tumor Cells via Bystander Effect</span><span
+
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style="color: windowtext; display: none; text-decoration: none;">
+
-
</span><span
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style="color: windowtext; display: none; text-decoration: none;">13</span></a></span></p>
+
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<p class="MsoToc3"><span class="MsoHyperlink"><a
+
-
href="#_Toc275981848"><span lang="EN-US">Conclusions</span><span
+
-
style="color: windowtext; display: none; text-decoration: none;">.
+
-
</span><span
+
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style="color: windowtext; display: none; text-decoration: none;">16</span></a></span></p>
+
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<p class="MsoToc3"><span class="MsoHyperlink"><a
+
-
href="#_Toc275981849"><span lang="EN-US">References</span><span
+
-
style="color: windowtext; display: none; text-decoration: none;">.
+
-
</span><span
+
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style="color: windowtext; display: none; text-decoration: none;">16</span></a></span></p>
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<p class="MsoNormal">&nbsp;</p>
<p class="MsoNormal">&nbsp;</p>
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<h2><a name="_Toc275981841"><span lang="EN-US">Arming:
+
 
-
Suicide Genes as GOIs</span></a></h2>
+
<h2><a name="_Toc275981841"><span lang="EN-US">Arming: Suicide Genes as GOIs</span></a></h2>
 +
 
<h3><a name="_Toc275981842"><span lang="EN-US">Introduction</span></a></h3>
<h3><a name="_Toc275981842"><span lang="EN-US">Introduction</span></a></h3>
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<p class="MsoNormal"><span lang="EN-US">Gene
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-
delivery using viral vectors to
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<p class="MsoNormal"><span lang="EN-US">Gene delivery using viral vectors to
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specifically target tumor cells gained increasing attention in the last
+
specifically target tumor cells gained increasing attention in the last years
-
years
+
being efficient in combination with suicide gene approaches </span><span lang="EN-US">(Willmon et al. 2006)</span><span lang="EN-US">. Several prodrug/enzyme
-
being efficient in combination with suicide gene approaches </span><span
+
combinations have been reported. Two systems - ganciclovir (GCV)/herpes simplex
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lang="EN-US">(Willmon et al. 2006)</span><span
+
virus thymidine kinase (HSV-TK) </span><span lang="EN-US">(Ardiani et al. 2010)</span><span lang="EN-US"> and
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lang="EN-US">. Several prodrug/enzyme
+
5-fluorocytosine/cytosine deaminase (CD) </span><span lang="EN-US">(Fuchita et al. 2009a)</span><span lang="EN-US"> – have been widely
-
combinations have been reported. The two systems - ganciclovir
+
used and their therapeutic benefit was demonstrated in preclinical studies </span><span lang="EN-US">(Greco &amp; Dachs 2001)</span><span lang="EN-US">. Adeno-associated
-
(GCV)/herpes
+
viruses (AAV) as delivery vectors are commonly used in suicide gene therapy. The
-
simplex virus thymidine kinase (HSV-TK) </span><span
+
suicide gene flanked by the inverted terminal repeats (ITRs) is encapsulated into
-
lang="EN-US">(Ardiani et al. 2010)</span><span
+
the virus particles and delivered to the target cells where suicide gene
-
lang="EN-US"> and
+
-
5-fluorocytosine/cytosine deaminase (CD) </span><span
+
-
lang="EN-US">(Fuchita et al. 2009a)</span><span
+
-
lang="EN-US"> – have been widely
+
-
used and their therapeutic benefit was demonstrated in preclinical
+
-
studies </span><span lang="EN-US">(Greco &amp;
+
-
Dachs 2001)</span><span lang="EN-US">.
+
-
Adeno-associated
+
-
viruses (AAV) as delivery vectors are commonly used in suicide gene
+
-
therapy. The
+
-
suicide gene flanked by the inverted terminal repeats (ITRs) is
+
-
encapsulated
+
-
into the virus particles and delivered to the target cells where
+
-
suicide gene
+
expression is mediated by cellular proteins.</span></p>
expression is mediated by cellular proteins.</span></p>
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<p class="MsoNormal"><span lang="EN-US">The
+
 
-
iGEM team Freiburg_Bioware 2010 provides
+
<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware 2010 provides
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both the cytosine deaminase (CD, BBa_K404112) and an improved guanylate
+
both the cytosine deaminase (CD, BBa_K404112) and an improved guanylate kinase -
-
kinase
+
thymidine kinase fusion gene (mGMK_TK, BBa_K404113) within the Virus
-
- thymidine kinase fusion gene (mGMK_TK, BBa_K404113) within the Virus
+
Construction Kit as effective suicide genes. We demonstrate efficient and
-
Construction Kit as effective suicide genes. We demonstrate efficient
+
-
and
+
specific killing of tumor cells by enzymatic cytotoxicity assays, flow
specific killing of tumor cells by enzymatic cytotoxicity assays, flow
-
cytometry, as well as phase contrast microscopy. HT1080 cancer cell
+
cytometry, as well as phase contrast microscopy. HT1080 cancer cell lines were
-
lines were
+
transduced with directed viral particles containing the suicide genes packaged
-
transduced with directed viral particles containing the suicide genes
+
-
packaged
+
into the viral capsids. </span></p>
into the viral capsids. </span></p>
-
<h3><a name="_Toc275981843"><span lang="EN-US">Successful
+
 
-
Assembly of Vector Plasmids
+
<h3><a name="_Toc275981843"><span lang="EN-US">Successful Assembly of Vector Plasmids
Carrying Suicide Genes via Cloning</span></a></h3>
Carrying Suicide Genes via Cloning</span></a></h3>
-
<p class="MsoNormal"><span lang="EN-US">Assembly
+
 
-
of the constructs carrying the
+
<p class="MsoNormal"><span lang="EN-US">To create the functional vector plasmids, assembly
-
suicide genes (termed vector plasmids) was performed following the
+
of the constructs carrying the suicide genes was performed following the
-
BioBrick
+
BioBrick Standard Assembly. All plasmids contain the enhancer-element human <i>beta-globin</i>
-
Standard Assembly. All plasmids contain the enhancer-element human <i>beta-globin</i>
+
intron (BBa_K404107) and the <i>human growth hormone</i> terminator signal (hGH,
-
intron (BBa_K404107) and the <i>human growth hormone</i>
+
BBa_K404108) flanked by the inverted terminal repeats (ITRs, BBa_K404100 and
-
terminator signal (hGH,
+
BBa_K404101). Assembled suicide genes are either under the control of the CMV
-
BBa_K404108) flanked by the inverted terminal repeats (ITRs,
+
promoter (BBa_J52034) or the tumor-specific telomerase promoter phTERT (BBa_K404106).
-
BBa_K404100 and
+
</span></p>
-
BBa_K404101). Assembled suicide genes are either under the control of
+
 
-
the CMV
+
-
promoter or the tumor-specific telomerase promoter phTERT
+
-
(BBa_K404106). </span></p>
+
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
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-
lang="EN-US">&nbsp;</span></p>
+
-
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
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<div align="center">
<div align="center">
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<table class="MsoTableGrid"
+
 
-
style="border: medium none ; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0">
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border="0" cellpadding="0" cellspacing="0">
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<tbody><tr style="height: 343.9pt;">
-
  <tbody>
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  <td style="padding: 0cm 5.4pt; width: 435.8pt; height: 343.9pt;" valign="top" width="581">
-
    <tr style="height: 343.9pt;">
+
  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 581px; height: 394px;" alt="" id="Grafik 9" src="http://2010.igem.org/wiki/images/9/9b/Freiburg10_BBA_overview_suicide.png"></p>
-
      <td
+
  <p class="MsoCaption"><a name="_Ref275976895"><span lang="EN-US">Figure </span></a><span lang="EN-US">1</span><span lang="EN-US">: BioBrick-compatible assembly of functional
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style="padding: 0cm 5.4pt; width: 435.8pt; height: 343.9pt;"
+
  vector plasmids containing the suicide genes. The schematic figure shows the
-
valign="top" width="581">
+
  cloning strategy of the guanylate kinase – thymidine kinase fusion gene
-
      <p class="MsoNormal"
+
  (mGMK_TK30).</span></p>
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style="text-indent: 0cm; page-break-after: avoid;"><img
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  </td>
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style="width: 573px; height: 388px;" alt="" id="Grafik 9"
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</tr>
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src="http://2010.igem.org/wiki/images/9/9b/Freiburg10_BBA_overview_suicide.png"></p>
+
</tbody></table>
-
      <p class="MsoCaption"><a name="_Ref275976895"><span
+
 
-
lang="EN-US">Figure </span></a><span
+
-
lang="EN-US">1</span><span lang="EN-US">:
+
-
BioBrick compatible assembly of functional
+
-
vector plasmids containing the suicide genes. The schematic figure
+
-
shows the
+
-
cloning strategy of the guanylate kinase – thymidine kinase fusion gene
+
-
(mGMK_TK30).</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
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<p class="MsoNormal" style="text-indent: 0cm;"><span
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lang="EN-US">&nbsp;</span></p>
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<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
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<p class="MsoNormal"><span lang="EN-US">In
+
 
-
order to modularize thymidine kinase
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<p class="MsoNormal"><span lang="EN-US">To enable modularization of the thymidine
-
mutants TK30 and SR39 (BBa_K404109 and BBa_K404110) according to the
+
kinase mutants TK30 and SR39 (BBa_K404109 and BBa_K404110) according to the
-
BioBrick
+
BioBrick standard, the fusion genes mGMK_TK30 and mGMK_SR39 (BBa_K404113 and </span><span style="line-height: 200%; color: black;" lang="EN-US">BBa_K404315</span><span lang="EN-US">) and CD (</span><span style="line-height: 200%; color: black;" lang="EN-US">BBa_K404112</span><span lang="EN-US">) were modified using the QuikChange
-
standard, the fusion genes mGMK_TK30 and mGMK_SR39 (BBa_K404113 and </span><span
+
Lightning Site-Directed Mutagenesis Kit (Stratagene) for deletion of iGEM RFC10
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style="line-height: 200%; color: black;" lang="EN-US">BBa_K404315</span><span
+
pre- and suffix restriction sites. </span><span lang="EN-US">Figure 1</span><span lang="EN-US"> demonstrates one example of
-
lang="EN-US">) and CD (</span><span
+
successful deletion of a PstI restriction site located within the mGMK_TK30
-
style="line-height: 200%; color: black;" lang="EN-US">BBa_K404112</span><span
+
sequence at position 3109. A point mutation was introduced replacing the
-
lang="EN-US">), were modified using the QuikChange
+
nucleotide G by A, resulting in the deletion of the restriction site while
-
Lightning Site-Directed Mutagenesis Kit (Stratagene) for deletion of
+
maintaining the encoded amino acid glutamine. Exchange of guanine to adenine
-
iGEM pre-
+
was confirmed by sequencing (</span><span lang="EN-US">Figure 2</span><span lang="EN-US">). </span></p>
-
and suffix restriction sites. </span><span lang="EN-US">Figure
+
 
-
1</span><span lang="EN-US"> demonstrates one example
+
-
of successful deletion of the PstI
+
-
restriction site located within the mGMK_TK30 sequence at position
+
-
3109. Base
+
-
pair exchange was introduced by replacing the nucleotide G with A,
+
-
resulting in
+
-
the deletion of the restriction site, but maintaining the amino acid
+
-
glutamine.
+
-
Successful transition of G to A was confirmed by sequencing (</span><span
+
-
lang="EN-US">Figure 2</span><span lang="EN-US">).
+
-
</span></p>
+
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
 
-
style="border: medium none ; width: 479.55pt; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; width: 479.55pt; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0" width="639">
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border="0" cellpadding="0" cellspacing="0"
+
<tbody><tr style="height: 179.55pt;">
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width="639">
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  <td style="padding: 0cm 5.4pt; width: 479.55pt; height: 179.55pt;" valign="top" width="639">
-
  <tbody>
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  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 635px; height: 328px;" alt="" id="Grafik 81" src="http://2010.igem.org/wiki/images/3/32/Freiburg10_SDM_mGMK_TK30.png"></p>
-
    <tr style="height: 179.55pt;">
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  <p class="MsoCaption"><a name="_Ref275976917"><span lang="EN-US">Figure </span></a><span lang="EN-US">2</span><span lang="EN-US">: Cytosine to guanine exchange by
-
      <td
+
  site-directed mutagenesis using QuikChange Lightning Kit provided by
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style="padding: 0cm 5.4pt; width: 479.55pt; height: 179.55pt;"
+
  Stratagene was successful as demonstrated by (A) test digestion linearizing
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valign="top" width="639">
+
  the plasmid with PstI and (B) by sequencing.</span></p>
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      <p class="MsoNormal"
+
  </td>
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style="text-indent: 0cm; page-break-after: avoid;"><img
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</tr>
-
style="width: 635px; height: 328px;" alt=""
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</tbody></table>
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id="Grafik 81"
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-
src="http://2010.igem.org/wiki/images/3/32/Freiburg10_SDM_mGMK_TK30.png"></p>
+
-
      <p class="MsoCaption"><a name="_Ref275976917"><span
+
-
lang="EN-US">Figure </span></a><span
+
-
lang="EN-US">2</span><span lang="EN-US">:
+
-
Replacing the nucleotide G with C by
+
-
site-directed mutagenesis using QuikChange Lightning Kit provided by
+
-
Stratagene has been successful performed as demonstrated by (A) test
+
-
digestion linearizing the plasmid with PstI and (B) by sequencing.</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
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</div>
</div>
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<p class="MsoNormal" style="text-indent: 0cm;"><span
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lang="EN-US">&nbsp;</span></p>
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<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
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<p class="MsoNormal"><span lang="EN-US">Furthermore,
+
 
-
assembly of BioBrick-compatible
+
<p class="MsoNormal"><span lang="EN-US">Furthermore, assembly of BioBrick-compatible
vector plasmids was performed. An example for the last assembly step of
vector plasmids was performed. An example for the last assembly step of
-
mGMK_TK30 and hGH_rITR is shown in </span><span lang="EN-US">Figure
+
mGMK_TK30 and hGH_rITR is shown in </span><span lang="EN-US">Figure 3</span><span lang="EN-US">. The plasmids were digested with
-
3</span><span lang="EN-US">. The plasmids were
+
both XbaI and PstI (New England Biolabs, Insert: BBa_K404116: hGH_rITR) or SpeI
-
digested with
+
and PstI (Vector) and loaded on an agarose gel. As demonstrated in the preparative
-
both XbaI and PstI (Insert: BBa_K404116: hGH_rITR) or SpeI and PstI
+
gel in </span><span lang="EN-US">Figure 3</span><span lang="EN-US">, the expected
-
(Vector) and
+
bands were detected under UV light and the extracted, subsequently purified DNA
-
loaded on an agarose gel. As demonstrated in the preparative gel in </span><span
+
was successfully ligated and transformed into <i>E. coli</i>. Each assembly
-
lang="EN-US">Figure 3</span><span lang="EN-US">,
+
step for producing the BioBricks was conducted following the iGEM BioBrick
-
the expected bands were detected
+
-
under UV light and the extracted DNA was be successfully ligated. Each
+
-
assembly
+
-
step for producing BioBricks was conducted following the iGEM BioBrick
+
standard.</span></p>
standard.</span></p>
 +
<div align="center">
<div align="center">
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<table class="MsoTableGrid"
+
 
-
style="border: medium none ; width: 387.35pt; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; width: 387.35pt; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0" width="516">
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border="0" cellpadding="0" cellspacing="0"
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<tbody><tr style="height: 125.95pt;">
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width="516">
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  <td style="padding: 0cm 5.4pt; width: 387.35pt; height: 125.95pt;" valign="top" width="516">
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  <tbody>
+
  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 519px; height: 189px;" alt="" id="Grafik 28" src="http://2010.igem.org/wiki/images/f/f2/Freiburg10_Cloning_mGMK_TK30_Arming.png"></p>
-
    <tr style="height: 125.95pt;">
+
  <p class="MsoCaption"><a name="_Ref275976959"><span lang="EN-US">Figure </span></a><span lang="EN-US">3</span><span lang="EN-US">: Assembly of mGMK_TK30 (vector molecule, in
-
      <td
+
  pSB1C3) and hGH-terminator_rightITR (insert molecule). The digested fragments
-
style="padding: 0cm 5.4pt; width: 387.35pt; height: 125.95pt;"
+
  were visualized under UV-light and correspond to the expected sizes.</span></p>
-
valign="top" width="516">
+
  </td>
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      <p class="MsoNormal"
+
</tr>
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style="text-indent: 0cm; page-break-after: avoid;"><img
+
</tbody></table>
-
style="width: 519px; height: 189px;" alt=""
+
 
-
id="Grafik 28"
+
-
src="http://2010.igem.org/wiki/images/f/f2/Freiburg10_Cloning_mGMK_TK30_Arming.png"></p>
+
-
      <p class="MsoCaption"><a name="_Ref275976959"><span
+
-
lang="EN-US">Figure </span></a><span
+
-
lang="EN-US">3</span><span lang="EN-US">:
+
-
Cloning of the composite parts mGMK_TK30
+
-
to hGH-terminator_rightITR (insert). The digested fragments correspond
+
-
to the
+
-
expected sizes.</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
 
-
lang="EN-US">&nbsp;</span></p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<h3><a name="_Toc275981844"><span lang="EN-US">Monitoring
+
 
-
Efficient Tumor Killing
+
<h3><a name="_Toc275981844"><span lang="EN-US">Monitoring Efficient Tumor Killing
by Phase-Contrast Microscopy</span></a></h3>
by Phase-Contrast Microscopy</span></a></h3>
-
<p class="MsoNormal"><span lang="EN-US">Tumor
+
 
-
cells, transduced with viral
+
<p class="MsoNormal"><span lang="EN-US">Tumor cells, transduced with viral
-
particles encapsidating the effector constructs containing the
+
particles encapsidating the effector constructs containing the mGMK_TK30 driven
-
mGMK_TK30 driven
+
by the CMV promoter, were cultured both in presence and absence of ganciclovir
-
by the CMV promoter, were cultured in presence and absence of
+
(Roche). Morphological changes were monitored via phase-contrast microscopy for
-
ganciclovir.
+
48 hours post infection. As it can be seen in </span><span lang="EN-US">Figure 4</span><span lang="EN-US">, non-transduced tumor cells treated
-
Morphological changes were monitored via phase-contrast microscopy
+
with ganciclovir and transduced cells without ganciclovir did not show
-
until 48
+
significant cell ablation. In contrast, transduced cells expressing the
-
hours post infection. As it can be seen in </span><span
+
guanylate kinase - thymidine kinase fusion protein showed significant cell
-
lang="EN-US">Figure 4</span><span lang="EN-US">
+
death after incubation with ganciclovir for 48 hours post infection. </span></p>
-
non-transduced cells treated with ganciclovir
+
 
-
and transduced cell without ganciclovir did not show significant tumor
+
<table class="MsoTableGrid" style="border: medium none ; width: 491pt; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0" width="655">
-
cell
+
<tbody><tr style="height: 188.8pt;">
-
ablation. In contrast transduced cells expressing the guanylate kinase
+
  <td style="padding: 0cm 5.4pt; width: 247.35pt; height: 188.8pt;" width="330">
-
-
+
  <p class="MsoNoSpacing"><b><span lang="EN-US">A</span></b><span lang="EN-US"> </span><img style="width: 318px; height: 238px;" id="Grafik 1" src="http://2010.igem.org/wiki/images/4/4f/Freiburg10_Microscopy_Arming_A.png" alt="Beschreibung: http://2010.igem.org/wiki/images/3/3e/HT_negativ_control_ganciclovir_only.jpg"></p>
-
thymidine kinase fusion protein, showed significant cell death after
+
  </td>
-
incubation
+
  <td style="padding: 0cm 5.4pt; width: 243.65pt; height: 188.8pt;" width="325">
-
with ganciclovir for 48 hours post infection. </span></p>
+
  <p class="MsoNoSpacing"><b><span lang="EN-US">B</span></b><span lang="EN-US"> </span><img style="width: 318px; height: 238px;" id="Grafik 2" src="http://2010.igem.org/wiki/images/e/e8/Freiburg10_Microscopy_Arming_B.png" alt="Beschreibung: http://2010.igem.org/wiki/images/f/f0/HT_TKGMK_clone_1_ohne_Ganciclovir_300%C2%B5l.jpg"></p>
-
<table class="MsoTableGrid"
+
  </td>
-
style="border: medium none ; width: 491pt; border-collapse: collapse;"
+
</tr>
-
border="0" cellpadding="0" cellspacing="0"
+
<tr style="height: 188.8pt;">
-
width="655">
+
  <td style="padding: 0cm 5.4pt; width: 247.35pt; height: 188.8pt;" width="330">
-
  <tbody>
+
  <p class="MsoNoSpacing"><b><span lang="EN-US">C</span></b><span lang="EN-US"> </span></p>
-
    <tr style="height: 188.8pt;">
+
  <p class="MsoNoSpacing" style="page-break-after: avoid;"><img style="width: 318px; height: 239px;" id="Grafik 3" src="http://2010.igem.org/wiki/images/a/ac/Freiburg10_Microscopy_Arming_C.png" alt="Beschreibung: http://2010.igem.org/wiki/images/c/c3/HT_TKGMK_clone_1_with_ganciclovir_300%C2%B5l.jpg"></p>
-
      <td
+
  </td>
-
style="padding: 0cm 5.4pt; width: 247.35pt; height: 188.8pt;"
+
  <td style="padding: 0cm 5.4pt; width: 243.65pt; height: 188.8pt;" width="325">
-
width="330">
+
  <p class="MsoNoSpacing"><b><span lang="EN-US">D</span></b><span lang="EN-US"> </span><img style="width: 318px; height: 238px;" id="Grafik 4" src="http://2010.igem.org/wiki/images/d/df/Freiburg10_Microscopy_Arming_D.png" alt="Beschreibung: http://2010.igem.org/wiki/images/7/73/HT_TKGMK_clone_1_with_ganciclovir_600%C2%B5l_well_2.jpg"></p>
-
      <p class="MsoNoSpacing"><b><span
+
  </td>
-
lang="EN-US">A</span></b><span lang="EN-US">
+
</tr>
-
      </span><img style="width: 318px; height: 238px;"
+
<tr style="height: 34.2pt;">
-
id="Grafik 1"
+
  <td colspan="2" style="padding: 0cm 5.4pt; width: 491pt; height: 34.2pt;" width="655">
-
src="http://2010.igem.org/wiki/images/4/4f/Freiburg10_Microscopy_Arming_A.png"
+
  <p class="MsoCaption"><a name="_Ref275976998"><span lang="EN-US">Figure </span></a><span lang="EN-US">4</span><span lang="EN-US">: Qualitative analysis of cell death induced
-
alt="Beschreibung: http://2010.igem.org/wiki/images/3/3e/HT_negativ_control_ganciclovir_only.jpg"></p>
+
  by conversion of ganciclovir to ganciclovir-triphosphate by virus-delivered
-
      </td>
+
  guanylate - thymidine kinase (mGMK_TK30). A: Non-transduced HT1080 cells
-
      <td
+
  incubated in the presence of ganciclovir not exhibiting cell death. B: Untreated
-
style="padding: 0cm 5.4pt; width: 243.65pt; height: 188.8pt;"
+
  transduced HT1080 cells showing a high survival rate. C: HT1080 cells
-
width="325">
+
  transduced with 300 µL viral particles and incubated with ganciclovir leading
-
      <p class="MsoNoSpacing"><b><span
+
  to tumor cell ablation. D: HT1080 cells transduced with 600 µL viral
-
lang="EN-US">B</span></b><span lang="EN-US">
+
  particles and incubated with ganciclovir leading to ablation of tumor cells. </span></p>
-
      </span><img style="width: 318px; height: 238px;"
+
  </td>
-
id="Grafik 2"
+
</tr>
-
src="http://2010.igem.org/wiki/images/e/e8/Freiburg10_Microscopy_Arming_B.png"
+
</tbody></table>
-
alt="Beschreibung: http://2010.igem.org/wiki/images/f/f0/HT_TKGMK_clone_1_ohne_Ganciclovir_300%C2%B5l.jpg"></p>
+
 
-
      </td>
+
-
    </tr>
+
-
    <tr style="height: 188.8pt;">
+
-
      <td
+
-
style="padding: 0cm 5.4pt; width: 247.35pt; height: 188.8pt;"
+
-
width="330">
+
-
      <p class="MsoNoSpacing"><b><span
+
-
lang="EN-US">C</span></b><span lang="EN-US">
+
-
      </span><img
+
-
style="width: 318px; height: 238px;" id="Grafik 3"
+
-
src="http://2010.igem.org/wiki/images/a/ac/Freiburg10_Microscopy_Arming_C.png"
+
-
alt="Beschreibung: http://2010.igem.org/wiki/images/c/c3/HT_TKGMK_clone_1_with_ganciclovir_300%C2%B5l.jpg"></p>
+
-
      </td>
+
-
      <td
+
-
style="padding: 0cm 5.4pt; width: 243.65pt; height: 188.8pt;"
+
-
width="325">
+
-
      <p class="MsoNoSpacing"><b><span
+
-
lang="EN-US">D</span></b><span lang="EN-US">
+
-
      </span><img style="width: 318px; height: 238px;"
+
-
id="Grafik 4"
+
-
src="http://2010.igem.org/wiki/images/d/df/Freiburg10_Microscopy_Arming_D.png"
+
-
alt="Beschreibung: http://2010.igem.org/wiki/images/7/73/HT_TKGMK_clone_1_with_ganciclovir_600%C2%B5l_well_2.jpg"></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr style="height: 34.2pt;">
+
-
      <td colspan="2"
+
-
style="padding: 0cm 5.4pt; width: 491pt; height: 34.2pt;"
+
-
width="655">
+
-
      <p class="MsoCaption"><a name="_Ref275976998"><span
+
-
lang="EN-US">Figure </span></a><span
+
-
lang="EN-US">4</span><span lang="EN-US">:
+
-
Qualitative analysis of cell death induced
+
-
by conversion of ganciclovir to ganciclovir-triphosphate by
+
-
virus-delivered
+
-
guanylate - thymidine kinase (mGMK_TK30). A: Non-transduced HT1080
+
-
cells
+
-
incubated in the presence of ganciclovir did not exhibit cell death. B:
+
-
Transduced HT1080 cells untreated resulting in survival of most cells.
+
-
C:
+
-
HT1080 cells were transduced with 300µL viral particles and incubated
+
-
with
+
-
ganciclovir leading to ablation of tumor cells. D: HT1080 cells were
+
-
transduced with 600µL viral particles and incubated with ganciclovir
+
-
leading
+
-
to ablation of tumor cells. </span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">Suicide
+
 
-
gene therapy is based on the
+
<p class="MsoNormal"><span lang="EN-US">Suicide gene therapy is based on the localized
-
conversion of non-toxic prodrugs to toxic substances </span><span
+
conversion of non-toxic prodrugs to toxic substances </span><span lang="EN-US">(Greco &amp; Dachs 2001)</span><span lang="EN-US">, promoting cell
-
lang="EN-US">(Greco &amp; Dachs 2001)</span><span
+
death in the tumor tissue (</span><span lang="EN-US">Figure 5</span><span lang="EN-US">). Directed gene delivery is achieved by using recombinant viral
-
lang="EN-US">, leading to cell
+
vectors as provided by the iGEM team Freiburg_Bioware 2010 within the Virus
-
death (</span><span lang="EN-US">Figure 5</span><span
+
Construction Kit.</span></p>
-
lang="EN-US">). Directed gene
+
 
-
delivery is achieved by using recombinant viral vectors as provided by
+
<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse; margin-left: 4.8pt; margin-right: 4.8pt;" align="left" border="0" cellpadding="0" cellspacing="0">
-
the iGEM
+
<tbody><tr style="height: 174.85pt;">
-
team Freiburg_Bioware 2010 within the Virus Construction Kit.</span></p>
+
  <td style="padding: 0cm 5.4pt; width: 447.8pt; height: 174.85pt;" valign="top" width="597">
-
<table class="MsoTableGrid"
+
  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><span style="position: relative; z-index: 251659264; left: -1px; top: 0px; width: 583px; height: 271px;"><img style="width: 597px; height: 279px;" alt="" src="http://2010.igem.org/wiki/images/a/a4/Freiburg10_overview_GDEPT.png"></span><br clear="all">
-
style="border: medium none ; border-collapse: collapse; margin-left: 4.8pt; margin-right: 4.8pt;"
+
  </p>
-
align="left" border="0" cellpadding="0"
+
  <p class="MsoCaption"><a name="_Ref275977093"><span lang="EN-US">Figure </span></a><span lang="EN-US">5</span><span lang="EN-US">: Overview of the suicide gene therapy
-
cellspacing="0">
+
  approach. Non-toxic prodrugs are converted into toxic effector molecules
-
  <tbody>
+
  leading to cell death of the tumor cells. </span></p>
-
    <tr style="height: 174.85pt;">
+
  </td>
-
      <td
+
</tr>
-
style="padding: 0cm 5.4pt; width: 447.8pt; height: 174.85pt;"
+
</tbody></table>
-
valign="top" width="597">
+
 
-
      <p class="MsoNormal"
+
<p class="MsoNormal"><span lang="EN-US">Non-transduced cells can survive in the
-
style="text-indent: 0cm; page-break-after: avoid;"><span
+
presence of ganciclovir since the prodrug is not toxic for these cells (</span><span lang="EN-US">Figure 4</span><span lang="EN-US">A). </span>The demonstration that
-
style="position: relative; z-index: 251658240; left: -1px; top: 0px; width: 583px; height: 271px;"><img
+
transduced cells are viable in the absence of ganciclovir confirms that cell
-
style="width: 583px; height: 271px;" alt=""
+
killing is indeed induced by combination of the delivered thymidine kinase and
-
src="http://2010.igem.org/wiki/images/a/a4/Freiburg10_overview_GDEPT.png"></span><br
+
treatment with ganciclovir. Viral particles encapsidating the suicide construct
-
clear="all">
+
mGMK_TK30 are efficient in directed gene delivery, thus leading to cell death
-
      </p>
+
of transduced cells due to overexpression of mGMK_TK30 and prodrug conversion. The
-
      <p class="MsoCaption"><a name="_Ref275977093"><span
+
cell-toxic ganciclovir-triphosphate is incorporated into the<span lang="EN-US">
-
lang="EN-US">Figure </span></a><span
+
nascent DNA chain leading to replication termination and finally resulting in
-
lang="EN-US">5</span><span lang="EN-US">:
+
death of dividing cells. </span></p>
-
Overview over the suicide gene therapy
+
 
-
approach. Non-toxic prodrugs are converted into toxic effector
+
<h3><a name="_Toc275981845"><span lang="EN-US">Quantitative Analysis of Cell
-
molecules
+
-
leading to cell death of the tumor cells. </span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
-
lang="EN-US">&nbsp;</span></p>
+
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
-
lang="EN-US">&nbsp;</span></p>
+
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
-
lang="EN-US">Since ganciclovir
+
-
is not toxic for cells, non-transduced cells can survive in the
+
-
presence of the
+
-
prodrug (</span><span lang="EN-US">Figure 4</span><span
+
-
lang="EN-US">A).
+
-
Demonstrating that transduced cells are viable in absence of
+
-
ganciclovir,
+
-
confirms that cell killing is induced by combination of delivered
+
-
thymidine
+
-
kinase and treatment with ganciclovir. Viral particles encapsidating
+
-
the
+
-
suicide construct mGMK_TK30 are efficient in directed gene delivery,
+
-
thus
+
-
leading to cell death of transduced cells due to overexpression of
+
-
mGMK_TK30
+
-
and prodrug conversion. The cell toxic ganciclovir-triphosphate is
+
-
incorporated
+
-
into the nascent DNA chain leading to replication termination and
+
-
finally
+
-
resulting in death of dividing cells. </span></p>
+
-
<h3><a name="_Toc275981845"><span lang="EN-US">Quantitative
+
-
Analysis of Cell
+
Death by Flow Cytometry</span></a></h3>
Death by Flow Cytometry</span></a></h3>
-
<p class="MsoNormal"><span lang="EN-US">Quantitative
+
 
-
analysis of the cytotoxic
+
<p class="MsoNormal"><span lang="EN-US">Quantitative analysis of the cytotoxic
-
effect induced by mGMK_TK30 was first conducted by flow cytometry
+
effect induced by mGMK_TK30 was first conducted by flow cytometry analysis 72 hours
-
analysis 72 hours
+
post transduction. HT1080 cells were stained with 7-AAD and Annexin V. 7-AAD intercalates
-
post transduction. HT1080 cells were stained with 7-AAD and Annexin V.
+
in double-stranded DNA after penetrating cell membranes of dead cells, whereas
-
7-AAD
+
Annexin V specifically binds phosphatidylserine which is only accessible during
-
intercalates in double-stranded DNA after penetrating cell membranes of
+
apoptosis. </span><span lang="EN-US">Figure 6</span><span lang="EN-US"> demonstrates
-
dead
+
the relation between cell death and ganciclovir concentration.</span></p>
-
cells, whereas Annexin V binds specifically phosphatidylserine which is
+
 
-
only
+
-
accessible during apoptosis. </span><span lang="EN-US">Figure
+
-
6</span><span lang="EN-US"> demonstrates the
+
-
relation between cell death and ganciclovir
+
-
concentration.</span></p>
+
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
 
-
style="border: medium none ; width: 477.6pt; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; width: 477.6pt; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0" width="637">
-
border="0" cellpadding="0" cellspacing="0"
+
<tbody><tr style="height: 248.5pt;">
-
width="637">
+
  <td style="padding: 0cm 5.4pt; width: 241.8pt; height: 248.5pt;" valign="top" width="322">
-
  <tbody>
+
  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 341px; height: 321px;" alt="" id="Grafik 24" src="http://2010.igem.org/wiki/images/9/9d/Freiburg10_FACS_A_D.png"></p>
-
    <tr style="height: 248.5pt;">
+
  </td>
-
      <td
+
  <td style="padding: 0cm 5.4pt; width: 235.8pt; height: 248.5pt;" valign="top" width="314">
-
style="padding: 0cm 5.4pt; width: 241.8pt; height: 248.5pt;"
+
  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 300px; height: 326px;" alt="" id="Grafik 36" src="http://2010.igem.org/wiki/images/0/0b/Freiburg10_FACS_E_H.png"></p>
-
valign="top" width="322">
+
  <p class="MsoCaption">&nbsp;</p>
-
      <p class="MsoNormal"
+
  </td>
-
style="text-indent: 0cm; page-break-after: avoid;"><img
+
</tr>
-
style="width: 308px; height: 326px;" alt=""
+
<tr style="height: 38pt;">
-
id="Grafik 24"
+
  <td colspan="2" style="padding: 0cm 5.4pt; width: 477.6pt; height: 38pt;" valign="top" width="637">
-
src="http://2010.igem.org/wiki/images/9/9d/Freiburg10_FACS_A_D.png"></p>
+
  <p class="MsoCaption" style="text-indent: 0cm;"><a name="_Ref275977177"><span lang="EN-US">Figure </span></a><span lang="EN-US">6</span><span lang="EN-US">: Flow
-
      </td>
+
  cytometry data analysis. </span><span lang="EN-US">A: Gating non-transduced
-
      <td
+
  HT1080 cells (control). B: Non-transduced cells without staining plotted
-
style="padding: 0cm 5.4pt; width: 235.8pt; height: 248.5pt;"
+
  against 7-AAD. C: Gating non-transduced cells stained with 7-AAD. D:
-
valign="top" width="314">
+
  Non-transduced, 7-AAD-stained cells plotted against 7-AAD. E: Gating
-
      <p class="MsoNormal"
+
  transduced cells (GOI: mGMK_TK30) treated with 485 µM ganciclovir. F: Gated,
-
style="text-indent: 0cm; page-break-after: avoid;"><img
+
  Annexin-V stained cells plotted against AnnV-2 Log.&nbsp; G: Gated cells&nbsp; plotted
-
style="width: 300px; height: 326px;" alt=""
+
  against 7-AAD H: Gated, 7-AAD and Annexin-V stained cells plotted against
-
id="Grafik 36"
+
  7-AAD and Annexin-V. Gate R19 comprised Annexin-V and 7-AAD positive cells.</span></p>
-
src="http://2010.igem.org/wiki/images/0/0b/Freiburg10_FACS_E_H.png"></p>
+
  </td>
-
      <p class="MsoCaption">&nbsp;</p>
+
</tr>
-
      </td>
+
</tbody></table>
-
    </tr>
+
 
-
    <tr style="height: 38pt;">
+
-
      <td colspan="2"
+
-
style="padding: 0cm 5.4pt; width: 477.6pt; height: 38pt;"
+
-
valign="top" width="637">
+
-
      <p class="MsoCaption" style="text-indent: 0cm;"><a
+
-
name="_Ref275977177"><span lang="EN-US">Figure </span></a><span
+
-
lang="EN-US">6</span><span lang="EN-US">:
+
-
      </span><span lang="EN-US">A: Gating
+
-
non-transduced HT1080 cells (control). B: Non-transduced
+
-
cells without staining plotted against 7-AAD. C: Gating non-transduced
+
-
cells
+
-
stained with 7-AAD. D: Non-transduced, 7-AAD-stained cells plotted
+
-
against
+
-
7-AAD. E: Gating transduced cells (GOI: mGMK_TK30) treated with 485µM
+
-
Ganciclovir. F: Gated, Annexin-V stained cells plotted against AnnV-2
+
-
Log. G:
+
-
Gated cells plotted against 7-AAD H: Gated, 7-AAD and Annexin-V stained
+
-
cells plotted against 7-AAD and Annexin-V. Gate R19 comprised Annexin-V
+
-
and
+
-
7-AAD positive cells.</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
 
-
lang="EN-US">&nbsp;</span></p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"
+
 
-
style="text-align: center; text-indent: 0cm;" align="center"><span
+
<p class="MsoNormal" style="text-align: center; text-indent: 0cm;" align="center"><span lang="EN-US">&nbsp;</span></p>
-
lang="EN-US">&nbsp;</span></p>
+
 
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
 
-
style="border: medium none ; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0">
-
border="0" cellpadding="0" cellspacing="0">
+
<tbody><tr>
-
  <tbody>
+
  <td style="padding: 0cm 5.4pt; width: 460.6pt;" valign="top" width="614">
-
    <tr>
+
  <p class="MsoNormal" style="text-align: center; text-indent: 0cm; page-break-after: avoid;" align="center"><img style="width: 614px; height: 424px;" alt="" id="Diagramm 8" src="http://2010.igem.org/wiki/images/2/29/Freiburg10_Diagram_mGMK_effectFACS.png"></p>
-
      <td style="padding: 0cm 5.4pt; width: 460.6pt;"
+
  <p class="MsoCaption"><a name="_Ref275977227"><span lang="EN-US">Figure </span></a><span lang="EN-US">7</span><span lang="EN-US">: Quantification of flow cytometry data
-
valign="top" width="614">
+
  provided in </span><span lang="EN-US">Figure 6</span><span lang="EN-US">. With
-
      <p class="MsoNormal"
+
  increasing ganciclovir concentration, the survival rate of cells decreases. 60
-
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+
  % of HT1080 cells treated with 4.85 mM ganciclovir show tumor ablation,
-
align="center"><img style="width: 562px; height: 387px;"
+
  however even lower amounts of ganciclovir lead to significant cell death.</span></p>
-
alt="" id="Diagramm 8"
+
  </td>
-
src="http://2010.igem.org/wiki/images/2/29/Freiburg10_Diagram_mGMK_effectFACS.png"></p>
+
</tr>
-
      <p class="MsoCaption"><a name="_Ref275977227"><span
+
</tbody></table>
-
lang="EN-US">Figure </span></a><span
+
 
-
lang="EN-US">7</span><span lang="EN-US">:
+
-
Quantification of flow cytometry data
+
-
provided in </span><span lang="EN-US">Figure 6</span><span
+
-
lang="EN-US">. With
+
-
increasing ganciclovir concentration, the survival rate of cells
+
-
decreases. 60%
+
-
of HT1080 cells treated with 4,85 mM ganciclovir show tumor ablation,
+
-
however
+
-
even lower amounts of ganciclovir led to significant cell death.</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
-
<p class="MsoNormal"
+
 
-
style="text-align: center; text-indent: 0cm;" align="center"><span
+
<p class="MsoNormal" style="text-align: center; text-indent: 0cm;" align="center"><span lang="EN-US">&nbsp;</span></p>
-
lang="EN-US">&nbsp;</span></p>
+
 
-
<p class="MsoNormal"><span lang="EN-US">Effect
+
<p class="MsoNormal"><span lang="EN-US">The effect of different ganciclovir
-
of different ganciclovir
+
concentrations on transduced HT1080 sarcoma cells was investigated.
-
concentrations on transduced HT1080 sarcoma cells. Transduction has
+
Transduction was performed with recombinant viral particles encapsidating the
-
been
+
mGMK_TK30 prodrug gene. 72 hours post infection, cells were stained with 7-AAD
-
performed with recombinant viral particles encapsidating the mGMK_TK30
+
and Annexin V. As </span><span lang="EN-US">Figure 7</span><span lang="EN-US"> shows,
-
prodrug
+
the fraction of killed transduced cells is proportional to the applied ganciclovir
-
gene. 72 hours post infection cells were stained with 7-AAD and Annexin
+
concentration.</span></p>
-
V. As </span><span lang="EN-US">Figure 7</span><span
+
 
-
lang="EN-US"> shows, the higher the ganciclovir
+
<h3><a name="_Toc275981846"><span lang="EN-US">Titrating Ganciclovir
-
concentration,
+
-
the more transduced cells were killed.</span></p>
+
-
<h3><a name="_Toc275981846"><span lang="EN-US">Titrating
+
-
Ganciclovir
+
Concentrations for Efficient Cell Killing by Cytotoxicity Assays</span></a></h3>
Concentrations for Efficient Cell Killing by Cytotoxicity Assays</span></a></h3>
-
<p class="MsoNormal"><span lang="EN-US">Further
+
 
-
analysis of the cytotoxic effect
+
<p class="MsoNormal"><span lang="EN-US">Further analysis of the cytotoxic effect
-
induced by thymidine kinase converting ganciclovir to the toxic
+
induced by thymidine kinase converting ganciclovir to the toxic anti-metabolite
-
anti-metabolite
+
has been performed using MTT assays. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium
-
has been performed using MTT assays.
+
bromide), also known as MTT, is a yellow-colored tetrazole, which is reduced to
-
3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium
+
purple insoluble formazan in the presence of NADH and NADPH </span><span lang="EN-US">(Roche n.d.)</span><span lang="EN-US">. Colorimetric analysis can be
-
bromide), also known as MTT, is a yellow tetrazole, which is reduced to
+
carried out via spectrometry. Different tumor cell lines, HT1080 and A431, were
-
purple insoluble
+
transduced with the recombinant viruses carrying the linear DNA construct
-
formazan in the presence of NADH and NADPH </span><span
+
coding for mGMK-TK30 regulated by the CMV promoter and subsequently treated
-
lang="EN-US">(Roche n.d.)</span><span lang="EN-US">.
+
with ganciclovir. 48 and 72 hours post infection, cells were incubated with MTT
-
The colorimetric analysis can be
+
and fresh DMEM. After cell lysis by DMSO, absorbance of formazan at 570 nm was
-
carried out via spectrometry. Different tumor cell lines, HT1080 and
+
quantified using a Tecan Sunrise plate reader. </span></p>
-
A431, were
+
 
-
transduced with the recombinant viruses carrying the linear DNA
+
-
construct
+
-
coding for mGMK-TK30 regulated by the CMV promoter and treated with
+
-
ganciclovir.
+
-
48 and 72 hours post infection cells were incubated with MTT and
+
-
absorbance of
+
-
formazan was quantified. </span></p>
+
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
 
-
style="border: medium none ; width: 451.05pt; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; width: 451.05pt; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0" width="601">
-
border="0" cellpadding="0" cellspacing="0"
+
<tbody><tr style="height: 176.7pt;">
-
width="601">
+
  <td style="padding: 0cm 5.4pt; width: 451.05pt; height: 176.7pt;" valign="top" width="601">
-
  <tbody>
+
  <p class="MsoNormal" style="text-indent: 0cm;"><b><span lang="EN-US">A</span></b><img style="width: 588px; height: 413px;" alt="" id="Diagramm 95" src="http://2010.igem.org/wiki/images/8/86/Freiburg10_MTT_72h_2d.png"></p>
-
    <tr style="height: 176.7pt;">
+
  </td>
-
      <td
+
</tr>
-
style="padding: 0cm 5.4pt; width: 451.05pt; height: 176.7pt;"
+
<tr style="height: 176.7pt;">
-
valign="top" width="601">
+
  <td style="padding: 0cm 3.5pt; width: 451.05pt; height: 176.7pt;" valign="top" width="601">
-
      <p class="MsoNormal" style="text-indent: 0cm;"><b><span
+
  <p class="MsoNormal" style="text-indent: 0cm;"><b>B</b> </p>
-
lang="EN-US">A</span></b><img
+
  <p class="MsoNormal" style="text-align: center; text-indent: 0cm; page-break-after: avoid;" align="center"><img style="width: 593px; height: 409px;" alt="" id="Diagramm 10" src="http://2010.igem.org/wiki/images/7/76/Freiburg10_MTT_72h_3d.png"></p>
-
style="width: 588px; height: 413px;" alt=""
+
  <p class="MsoCaption"><a name="_Ref275977309"><span lang="EN-US">Figure </span></a><span lang="EN-US">8</span><span lang="EN-US">: Effect of ganciclovir on HT1080 cell survival
-
id="Diagramm 95"
+
  72 hours post infection as (A) two dimensional plot of survival of cells and
-
src="http://2010.igem.org/wiki/images/8/86/Freiburg10_MTT_72h_2d.png"></p>
+
  (B) three-dimensional plot of ganciclovir, virus particles and cell survival.</span></p>
-
      </td>
+
  </td>
-
    </tr>
+
</tr>
-
    <tr style="height: 176.7pt;">
+
</tbody></table>
-
      <td
+
 
-
style="padding: 0cm 3.5pt; width: 451.05pt; height: 176.7pt;"
+
-
valign="top" width="601">
+
-
      <p class="MsoNormal" style="text-indent: 0cm;"><b>B</b>
+
-
      </p>
+
-
      <p class="MsoNormal"
+
-
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+
-
align="center"><img style="width: 540px; height: 372px;"
+
-
alt="" id="Diagramm 10"
+
-
src="http://2010.igem.org/wiki/images/7/76/Freiburg10_MTT_72h_3d.png"></p>
+
-
      <p class="MsoCaption"><a name="_Ref275977309"><span
+
-
lang="EN-US">Figure </span></a><span
+
-
lang="EN-US">8</span><span lang="EN-US">:
+
-
Effect of ganciclovir on HT1080 cell
+
-
killing 72 hours post infection as (A) two dimensional plot of survival
+
-
of
+
-
cells and (B) three-dimensional plot of ganciclovir, virus particles
+
-
and cell
+
-
survival.</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
 
-
style="font-size: 8pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span style="font-size: 8pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
 
-
style="font-size: 8pt; line-height: 200%;" lang="EN-US"></span></p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span style="font-size: 8pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
 +
 
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span style="font-size: 8pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
 +
 
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
 
-
style="border: medium none ; width: 415pt; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; width: 415pt; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0" width="553">
-
border="0" cellpadding="0" cellspacing="0"
+
<tbody><tr style="height: 176.7pt;">
-
width="553">
+
  <td style="padding: 0cm 5.4pt; width: 415pt; height: 176.7pt;" valign="top" width="553">
-
  <tbody>
+
  <p class="MsoNormal" style="text-indent: 0cm;"><b><span lang="EN-US">A </span></b><span lang="EN-US">&nbsp;</span></p>
-
    <tr style="height: 176.7pt;">
+
  <p class="MsoNormal" style="text-align: center; text-indent: 0cm;" align="center"><img style="width: 607px; height: 427px;" alt="" id="Diagramm 1154" src="http://2010.igem.org/wiki/images/2/2a/Freiburg10_MTT_96h_2d.png"></p>
-
      <td style="padding: 0cm 5.4pt; width: 415pt; height: 176.7pt;"
+
  </td>
-
valign="top" width="553">
+
</tr>
-
      <p class="MsoNormal" style="text-indent: 0cm;"><b><span
+
<tr style="height: 176.7pt;">
-
lang="EN-US">A </span></b><span lang="EN-US">
+
  <td style="padding: 0cm 5.4pt; width: 415pt; height: 176.7pt;" valign="top" width="553">
-
      </span></p>
+
  <p class="MsoNormal" style="text-indent: 0cm;"><b>B</b></p>
-
      <p class="MsoNormal"
+
  <p class="MsoNormal" style="text-align: center; text-indent: 0cm; page-break-after: avoid;" align="center"><img style="width: 607px; height: 481px;" alt="" id="Diagramm 1156" src="http://2010.igem.org/wiki/images/0/0a/Freiburg10_MTT_96h_3d.png"></p>
-
style="text-align: center; text-indent: 0cm;" align="center"><img
+
  <p class="MsoCaption"><a name="_Ref275977314"><span lang="EN-US">Figure </span></a><span lang="EN-US">9</span><span lang="EN-US">: Effect of ganciclovir on HT1080 cell survival
-
style="width: 607px; height: 427px;" alt=""
+
  96 hours post infection. (A) two dimensional plot of cell survival&nbsp; (B)
-
id="Diagramm 1154"
+
  three-dimensional plot of ganciclovir, virus particles and cell survival.</span></p>
-
src="http://2010.igem.org/wiki/images/2/2a/Freiburg10_MTT_96h_2d.png"></p>
+
  </td>
-
      </td>
+
</tr>
-
    </tr>
+
</tbody></table>
-
    <tr style="height: 176.7pt;">
+
 
-
      <td style="padding: 0cm 5.4pt; width: 415pt; height: 176.7pt;"
+
-
valign="top" width="553">
+
-
      <p class="MsoNormal" style="text-indent: 0cm;"><b>B</b></p>
+
-
      <p class="MsoNormal"
+
-
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+
-
align="center"><img style="width: 606px; height: 480px;"
+
-
alt="" id="Diagramm 1156"
+
-
src="http://2010.igem.org/wiki/images/0/0a/Freiburg10_MTT_96h_3d.png"></p>
+
-
      <p class="MsoCaption"><a name="_Ref275977314"><span
+
-
lang="EN-US">Figure </span></a><span
+
-
lang="EN-US">9</span><span lang="EN-US">:
+
-
Effect of ganciclovir on HT1080 cell
+
-
killing 96 hours post infection as (A) two dimensional plot of survival
+
-
of
+
-
cells and (B) three-dimensional plot of ganciclovir, virus particles
+
-
and cell
+
-
survival.</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
 
-
style="font-size: 8pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span style="font-size: 8pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">Data
+
 
-
of MTT assay quantification are shown
+
<p class="MsoNormal"><span lang="EN-US">Data of MTT assay quantification are shown
-
in </span><span lang="EN-US">Figure 8</span><span
+
in </span><span lang="EN-US">Figure 8</span><span lang="EN-US"> and </span><span lang="EN-US">Figure 9</span><span lang="EN-US">. HT1080 cells were infected with
-
lang="EN-US"> and </span><span lang="EN-US">Figure
+
viral particles containing the mGMK_TK30 transgene. &nbsp;72 h- and 96 h post
-
9</span><span lang="EN-US">. HT1080 cells were
+
infection and addition of ganciclovir, cells were incubated with MTT. Changes
-
infected with
+
in absorbance were measured and survival of cells plotted against ganciclovir
-
viral particles containing the mGMK_TK30 transgene. 72 h- and 96 h post
+
concentration. </span><span lang="EN-US">Figure 8</span><span lang="EN-US">A
-
infection and addition of ganciclovir, cells were incubated with MTT.
+
demonstrates the correlation between increasing ganciclovir concentrations and
-
Changes
+
-
in absorbance were measured and survival of cells plotted against
+
-
ganciclovir
+
-
concentration. </span><span lang="EN-US">Figure 8</span><span
+
-
lang="EN-US">A
+
-
demonstrates the correlation between increasing ganciclovir
+
-
concentrations and
+
percentage of cell survival. Furthermore, different virus particle
percentage of cell survival. Furthermore, different virus particle
-
concentrations were used for transduction. </span><span
+
concentrations were used for transduction. </span><span lang="EN-US">Figure 8</span><span lang="EN-US">B shows that the highest amount of
-
lang="EN-US">Figure 8</span><span lang="EN-US">B
+
viral particles combined with the highest ganciclovir concentration led to
-
shows that the highest amount of
+
-
viral particles combined with the highest ganciclovir concentration led
+
-
to
+
significant HT1080 apoptosis 72 hours post transduction.</span></p>
significant HT1080 apoptosis 72 hours post transduction.</span></p>
-
<p class="MsoNormal"><span lang="EN-US">Additionally
+
 
-
96 hours post infection cells
+
<p class="MsoNormal"><span lang="EN-US">Additionally, 96 hours post infection cells
-
were incubated with MTT and absorbance was quantified via spectrometry (</span><span
+
were incubated with MTT and absorbance was quantified via spectrometry (</span><span lang="EN-US">Figure 9</span><span lang="EN-US">). Again, survival of HT1080 cells
-
lang="EN-US">Figure 9</span><span lang="EN-US">).
+
-
Again, survival of HT1080 cells
+
was plotted against increasing ganciclovir concentrations. </span></p>
was plotted against increasing ganciclovir concentrations. </span></p>
-
<h3><a name="_Toc275981847"><span lang="EN-US">Killing
+
 
-
Untransduced Tumor Cells
+
<h3><a name="_Toc275981847"><span lang="EN-US">Killing Non-transduced Tumor Cells
-
via Bystander Effect</span></a><span lang="EN-US">
+
via Bystander Effect</span></a><span lang="EN-US"> </span></h3>
-
</span></h3>
+
 
-
<p class="MsoNormal"><span lang="EN-US">The
+
<p class="MsoNormal"><span lang="EN-US">The bystander effect was first reported by </span><span lang="EN-US">Moolten (1986)</span><span lang="EN-US"> showing that prodrug
-
bystander effect was first reported by </span><span
+
convertase negative cells surrounded by suicide enzyme positive cells did not
-
lang="EN-US">Moolten (1986)</span><span lang="EN-US">
+
survive prodrug treatment. Besides efficient killing of targeted tumor cells,
-
showing that prodrug
+
neighboring, non-transduced cells are killed as well, providing an important
-
convertase negative cells surrounded by suicide enzyme positive cells
+
effect in cancer treatment. Since 5-Fluorouracil is soluble and can diffuse
-
did not
+
into adjacent cells </span><span lang="EN-US">(Huber et al. 1993)</span><span lang="EN-US"> </span><span lang="EN-US">(Huber et al. 1994)</span><span lang="EN-US">, the bystander effect
-
survive prodrug treatment. Besides efficient killing of targeted tumor
+
-
cells,
+
-
neighboring, non-transduced cells are killed as well, providing an
+
-
important
+
-
effect in treating cancer. Since 5-Fluorouracil is soluble and can
+
-
diffuse into
+
-
adjacent cells </span><span lang="EN-US">(Huber et
+
-
al. 1993)</span><span lang="EN-US"> </span><span
+
-
lang="EN-US">(Huber et al. 1994)</span><span
+
-
lang="EN-US">, the bystander effect
+
was demonstrated using cytosine deaminase as gene of interest.</span></p>
was demonstrated using cytosine deaminase as gene of interest.</span></p>
 +
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
 +
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
 
-
style="border: medium none ; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0">
-
border="0" cellpadding="0" cellspacing="0">
+
<tbody><tr>
-
  <tbody>
+
  <td style="padding: 0cm 5.4pt; width: 460.6pt;" valign="top" width="614">
-
    <tr>
+
  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 614px; height: 262px;" alt="" id="Grafik 82" src="http://2010.igem.org/wiki/images/c/c0/Freiburg10_Bystander_effect.png"></p>
-
      <td style="padding: 0cm 5.4pt; width: 460.6pt;"
+
  <p class="MsoCaption"><span lang="EN-US">Figure </span><span lang="EN-US">10</span><span lang="EN-US">: Schematic overview of the Bystander
-
valign="top" width="614">
+
  effect.</span></p>
-
      <p class="MsoNormal"
+
  </td>
-
style="text-indent: 0cm; page-break-after: avoid;"><img
+
</tr>
-
style="width: 605px; height: 262px;" alt=""
+
</tbody></table>
-
id="Grafik 82"
+
 
-
src="http://2010.igem.org/wiki/images/c/c0/Freiburg10_Bystander_effect.png"></p>
+
-
      <p class="MsoCaption"><span lang="EN-US">Figure
+
-
      </span><span lang="EN-US">10</span><span
+
-
lang="EN-US">: Schematic overview of the Bystander
+
-
effect.</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
 +
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">The
+
 
-
aim was to investigate if the modified
+
<p class="MsoNormal"><span lang="EN-US">The aim was to investigate if the modified
-
AAV-2 is able to kill tumor cells with the cytosine deaminase (CD) as
+
AAV-2 is able to kill tumor cells with the cytosine deaminase (CD) as gene of interest.
-
gene of interest.
+
The viral particles were produced according to the standard protocol using the
-
The viral particles were produced according to the standard protocol
+
-
using the
+
following plasmids:</span></p>
following plasmids:</span></p>
-
<p class="MsoListParagraphCxSpFirst"
+
 
-
style="margin-left: 53.85pt; text-indent: -18pt;"><span
+
<p class="MsoListParagraphCxSpFirst" style="margin-left: 53.85pt; text-indent: -18pt;"><span style="font-family: Symbol;">·<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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
style="font-family: Symbol;">·<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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
</span></span>pHelper</p>
</span></span>pHelper</p>
-
<p class="MsoListParagraphCxSpMiddle"
+
 
-
style="margin-left: 53.85pt; text-indent: -18pt;"><span
+
<p class="MsoListParagraphCxSpMiddle" style="margin-left: 53.85pt; text-indent: -18pt;"><span style="font-family: Symbol;" 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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
style="font-family: Symbol;" 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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
</span></span><span lang="EN-US">Rep/Cap:
</span></span><span lang="EN-US">Rep/Cap:
pSB1C3_001_[AAV2]-Rep-VP123(ViralBrick-587KO-empty)_p5-TATAless</span></p>
pSB1C3_001_[AAV2]-Rep-VP123(ViralBrick-587KO-empty)_p5-TATAless</span></p>
-
<p class="MsoListParagraphCxSpLast"
+
 
-
style="margin-left: 53.85pt; text-indent: -18pt;"><span
+
<p class="MsoListParagraphCxSpLast" style="margin-left: 53.85pt; text-indent: -18pt;"><span style="font-family: Symbol;" 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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
style="font-family: Symbol;" lang="EN-US">·<span
+
</span></span><span lang="EN-US">Gene of interest:
-
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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
-
</span></span><span lang="EN-US">Gene of
+
-
interest:
+
pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_CD_hGH_[AAV2]-right-ITR</span></p>
pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_CD_hGH_[AAV2]-right-ITR</span></p>
-
<p class="MsoNormal"><span lang="EN-US">90 %
+
 
-
confluent HT1080 cells were transduced
+
<p class="MsoNormal"><span lang="EN-US">90 % confluent HT1080 cells were transduced
-
in T75 flasks with 9 ml of viral stock. In parallel, another T75 flask
+
in T75 flasks with 9 ml of viral stock. In parallel, another T75 flask was
-
was
+
transduced with a viral stock packaged with the mVenus coding sequence to
-
transduced with a viral stock packaged with mVenus to assess transgene
+
assess transgene expression.</span></p>
-
expression.</span></p>
+
 
-
<p class="MsoNormal"><span lang="EN-US">24
+
<p class="MsoNormal"><span lang="EN-US">24 hours before harvesting the
-
hours before harvesting the
+
CD-transduced cells, two six well plates with 200.000 cells per well were
-
CD-transduced cells, two six well plates with 200.000 cells per well
+
seeded. After 30 hours, mVenus expression was observed and the CD-transduced
-
were
+
HT1080 were harvested. To minimize the influence of viral particles in the
-
seeded. After 30 hours, mVenus expression was observed and the
+
medium, the cells were washed four times with PBS. The cells were counted via a
-
CD-transduced
+
Neubauer cell chamber and 100.000 cells per well of a six well plate were
-
HT1080 were harvested. To minimize the influence of viral particles in
+
seeded. Additionally, 100.000 of the CD-transduced cells were seeded onto previously
-
the
+
-
medium, the cells were washed four times with PBS. The cells were
+
-
counted via a
+
-
Neubauer cell chamber and 100.000 cells per well of a six well plate
+
-
were
+
-
seeded. Additionally, 100.000 of the CD-transduced cells were seeded
+
-
onto previously
+
seeded untransduced HT1080.</span></p>
seeded untransduced HT1080.</span></p>
-
<p class="MsoNormal"><span lang="EN-US">The
+
 
-
incubation with the prodrug
+
<p class="MsoNormal"><span lang="EN-US">The incubation with the prodrug
-
5-fluorocytosine (5-FC) was performed at a final concentration of 53
+
5-fluorocytosine (5-FC) was performed at a final concentration of 53 mM.
-
mM.
+
According to Fuchita <i>et al.,</i> this amount should be sufficient to show
-
According to Fuchita <i>et al.,</i> this amount should be
+
the functionality of the CD </span><span lang="EN-US">(Fuchita et al. 2009b)</span><span lang="EN-US">. As demonstrated in </span><span lang="EN-US">Figure 11</span><span lang="EN-US">, cytotoxicity of 5-FC is
-
sufficient to show
+
remarkable.</span></p>
-
the functionality of the CD </span><span lang="EN-US">(Fuchita
+
 
-
et al. 2009b)</span><span lang="EN-US">. As
+
-
demonstrated in </span><span lang="EN-US">Figure 11</span><span
+
-
lang="EN-US"> cytotoxicity of 5-FC is remarkable.</span></p>
+
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
 
-
style="border: medium none ; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0">
-
border="0" cellpadding="0" cellspacing="0">
+
<tbody><tr style="height: 351pt;">
-
  <tbody>
+
  <td style="padding: 0cm 5.4pt; width: 457.65pt; height: 351pt;" valign="top" width="610">
-
    <tr style="height: 351pt;">
+
  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 610px; height: 424px;" alt="" src="http://2010.igem.org/wiki/images/4/4c/Freiburg10_CD_ToxicEffect.png" id="Diagramm 5"></p>
-
      <td style="padding: 0cm 5.4pt; width: 457.65pt; height: 351pt;"
+
  <p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure </span><span lang="EN-US">11</span><span lang="EN-US">: </span><span style="font-weight: normal;" lang="EN-US">Transduction of HT1080 with cytosine deaminase-packed viral
-
valign="top" width="610">
+
  particles. 5-FC: 5-fluorocytosine (53 mM)</span></p>
-
      <p class="MsoNormal"
+
  </td>
-
style="text-indent: 0cm; page-break-after: avoid;"><img
+
</tr>
-
style="width: 606px; height: 421px;" alt=""
+
</tbody></table>
-
id="Diagramm 5"
+
 
-
src="http://2010.igem.org/wiki/images/4/4c/Freiburg10_CD_ToxicEffect.png"></p>
+
-
      <p class="MsoCaption" style="text-indent: 0cm;"><span
+
-
lang="EN-US">Figure </span><span lang="EN-US">11</span><span
+
-
lang="EN-US">: </span><span
+
-
style="font-weight: normal;" lang="EN-US">Transduction
+
-
of HT1080 with cytosine deaminase-packed viral
+
-
particles. 5-FC: 5-fluorocytosine (53 mM)</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
-
<p class="MsoNormal" style="text-indent: 0cm;"><span
+
 
-
lang="EN-US">&nbsp;</span></p>
+
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">After
+
 
-
three days of incubation in 5-fluorocytosine,
+
<p class="MsoNormal"><span lang="EN-US">After three days of incubation in 5-fluorocytosine,
-
the cells were washed, detached with Trypsin, centrifuged at 200 g for
+
the cells were washed, detached with Trypsin, centrifuged at 200 g for 5 min
-
5 min
+
followed by two washing steps with PBS and finally resuspended with 200 µl
-
followed by two washing steps with PBS and finally resuspended with 200
+
-
µl
+
DMEM. Living cells were then counted via Trypan blue staining.</span></p>
DMEM. Living cells were then counted via Trypan blue staining.</span></p>
-
<p class="MsoNormal"><span lang="EN-US">After
+
 
-
this successful qualitative demonstration
+
<p class="MsoNormal"><span lang="EN-US">After this successful qualitative
-
of an AAV2-mediated cytosine deaminase treatment, the bystander effect
+
demonstration of an AAV2-mediated cytosine deaminase treatment, the bystander
-
was quantified
+
effect was quantified as well. The activated 5-FC molecules are able to diffuse
-
as well. The activated 5-FC molecules are able to diffuse through the
+
through the plasma membrane and thus effect cells that are not transduced (</span><span lang="EN-US">Figure 12</span><span lang="EN-US">). The bystander effect was tested with
-
plasma
+
untransduced HT1080 cells, which were mixed with the CD-transduced HT1080 cells</span><span lang="EN-US">.</span></p>
-
membrane and effect cells that are not transduced (</span><span
+
 
-
lang="EN-US">Figure 12</span><span lang="EN-US">).
+
-
The bystander effect was tested with
+
-
untransduced HT1080 cells, which were mixed with the CD-transduced
+
-
HT1080 cells</span><span lang="EN-US">.</span></p>
+
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
 
-
style="border: medium none ; border-collapse: collapse;"
+
<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0">
-
border="0" cellpadding="0" cellspacing="0">
+
<tbody><tr>
-
  <tbody>
+
  <td style="padding: 0cm 5.4pt; width: 460.6pt;" valign="top" width="614">
-
    <tr>
+
  <p class="MsoNormal" style="text-align: left; text-indent: 0cm; page-break-after: avoid;" align="left"><img style="width: 614px; height: 483px;" alt="" id="Diagramm 1158" src="http://2010.igem.org/wiki/images/8/8c/Freiburg10_CD_Bystander.png"></p>
-
      <td style="padding: 0cm 5.4pt; width: 460.6pt;"
+
  <p class="MsoCaption" style="text-align: left;" align="left"><a name="_Ref275979941"><span lang="EN-US">Figure </span></a><span lang="EN-US">12</span><span lang="EN-US">: </span><span style="font-weight: normal;" lang="EN-US">Quantifying the bystander effect on
-
valign="top" width="614">
+
  HT1080 cells&nbsp; 5-FC: 5-fluorocytosine (53 mM)</span></p>
-
      <p class="MsoNormal"
+
  </td>
-
style="text-align: left; text-indent: 0cm; page-break-after: avoid;"
+
</tr>
-
align="left"><img style="width: 607px; height: 478px;"
+
</tbody></table>
-
alt="" id="Diagramm 1158"
+
 
-
src="http://2010.igem.org/wiki/images/8/8c/Freiburg10_CD_Bystander.png"></p>
+
-
      <p class="MsoCaption" style="text-align: left;"
+
-
align="left"><a name="_Ref275979941"><span
+
-
lang="EN-US">Figure </span></a><span
+
-
lang="EN-US">12</span><span lang="EN-US">:
+
-
      </span><span style="font-weight: normal;"
+
-
lang="EN-US">Quantifying the bystander effect on
+
-
HT1080 cells 5-FC: 5-fluorocytosine (53 mM)</span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
</div>
</div>
 +
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">The
+
 
-
cytosine deaminase expressing cells
+
<p class="MsoNormal"><span lang="EN-US">The cytosine deaminase expressing cells
-
have an obvious effect on the viability of the non-transduced cells. As
+
have an obvious effect on the viability of the non-transduced cells. As shown
-
shown
+
in the graph we were able to demonstrate that cytosine deaminase mediated cancer
-
in the graph we were able to demonstrate that cytosine deaminase
+
-
mediated cancer
+
cell death is also fatal for non-transduced cells in close proximity.</span></p>
cell death is also fatal for non-transduced cells in close proximity.</span></p>
 +
<h3><a name="_Toc275981848"><span lang="EN-US">Conclusions</span></a></h3>
<h3><a name="_Toc275981848"><span lang="EN-US">Conclusions</span></a></h3>
-
<p class="MsoNormal"><span lang="EN-US">Efficient
+
 
-
and tissue-specific tumor killing
+
<p class="MsoNormal"><span lang="EN-US">Efficient and tissue-specific tumor killing
-
is one major challenge in cancer therapy </span><span
+
is one major challenge in cancer therapy </span><span lang="EN-US">(Black et al. 1996)</span><span lang="EN-US">. Gene-directed enzyme
-
lang="EN-US">(Black et al. 1996)</span><span
+
prodrug therapy (GDEPT) is based on the conversion of non-toxic substances into
-
lang="EN-US">. Gene-directed enzyme
+
toxic drugs resulting in tumor cell death. The iGEM team Freiburg_Bioware 2010
-
prodrug therapy (GDEPT) is based on the conversion of non-toxic
+
provides several functional suicide genes within the Virus Construction Kit, thus
-
substances into
+
offering a feasible and modular tool to the growing field of personalized
-
toxic drugs resulting in tumor cell death. The iGEM team
+
medicine and the iGEM community. We successfully demonstrated cancer cell death
-
Freiburg_Bioware 2010
+
caused by the introduction of cytosine deaminase and modified fusion genes
-
provides several functional suicide genes within the Virus Construction
+
-
Kit. Thus
+
-
offering a feasible and modular tool to the growing field of
+
-
personalized
+
-
medicine and the iGEM community. We successfully demonstrated cancer
+
-
cell death
+
-
caused by the introduction of cytosine deaminase and modified fusion
+
-
genes
+
consisting of guanylate and thymidine kinases.</span></p>
consisting of guanylate and thymidine kinases.</span></p>
-
<p class="MsoNormal"><span lang="EN-US"> To
+
 
-
prevent systemic toxic side effects of
+
<p class="MsoNormal"><span lang="EN-US">&nbsp;To prevent systemic toxic side effects of
-
conventional chemotherapy the iGEM team Freiburg_Bioware 2010 took a
+
conventional chemotherapy, the iGEM team Freiburg_Bioware 2010 took a leap and
-
leap and
+
efficiently retargeted the viral vector for directed suicide gene delivery
-
efficiently retargeted the viral vector for directed suicide gene
+
towards tumor cells. Capsid engineering was successfully demonstrated by the
-
delivery
+
iGEM team Freiburg_Bioware 2010. Further details can be found under Results –
-
towards tumor cells. Capsid engineering was successfully demonstrated
+
-
by the
+
-
iGEM team Freiburg_Bioware 2010. Further details can be found under
+
-
Results –
+
Targeting.</span></p>
Targeting.</span></p>
 +
<h3><a name="_Toc275981849"><span lang="EN-US">References</span></a></h3>
<h3><a name="_Toc275981849"><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;;"
+
<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Ardiani, A., Sanchez-Bonilla, M. &amp;
-
lang="EN-US">Ardiani, A., Sanchez-Bonilla, M. &amp;
+
Black, M.E., 2010. Fusion enzymes containing HSV-1 thymidine kinase mutants and
-
Black, M.E., 2010. Fusion enzymes containing HSV-1 thymidine kinase
+
-
mutants and
+
guanylate kinase enhance prodrug sensitivity in vitro and in vivo. <i>Cancer
guanylate kinase enhance prodrug sensitivity in vitro and in vivo. <i>Cancer
gene therapy</i>, 17(2), 86-96. Available at:
gene therapy</i>, 17(2), 86-96. Available at:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2808426&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2808426&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
-
<p style="text-indent: 36pt;"><span
+
 
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Black, M.E. et al., 1996. Creation of
-
lang="EN-US">Black, M.E. et al., 1996. Creation of
+
drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene
-
drug-specific herpes simplex virus type 1 thymidine kinase mutants for
+
therapy. <i>Proceedings of the National Academy of Sciences of the United
-
gene
+
-
therapy. <i>Proceedings of the National Academy of Sciences of
+
-
the United
+
States of America</i>, 93(8), 3525-9. Available at:
States of America</i>, 93(8), 3525-9. Available at:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=39643&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=39643&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
-
<p style="text-indent: 36pt;"><span
+
 
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Fuchita, M. et al., 2009. Bacterial
-
lang="EN-US">Fuchita, M. et al., 2009. Bacterial
+
cytosine deaminase mutants created by molecular engineering show improved
-
cytosine deaminase mutants created by molecular engineering show
+
5-fluorocytosine-mediated cell killing in vitro and in vivo. <i>Cancer research</i>,
-
improved
+
-
5-fluorocytosine-mediated cell killing in vitro and in vivo. <i>Cancer
+
-
research</i>,
+
69(11), 4791-9. Available at:
69(11), 4791-9. Available at:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2765227&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2765227&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
-
<p style="text-indent: 36pt;"><span
+
 
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Fuchita, M. et al., 2009. Bacterial
-
lang="EN-US">Fuchita, M. et al., 2009. Bacterial
+
cytosine deaminase mutants created by molecular engineering show improved
-
cytosine deaminase mutants created by molecular engineering show
+
5-fluorocytosine-mediated cell killing in vitro and in vivo. <i>Cancer research</i>,
-
improved
+
-
5-fluorocytosine-mediated cell killing in vitro and in vivo. <i>Cancer
+
-
research</i>,
+
69(11), 4791-9. Available at:
69(11), 4791-9. Available at:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2765227&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2765227&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
-
<p style="text-indent: 36pt;"><span
+
 
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Greco,
+
<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Greco,
-
O. &amp; Dachs, G.U., 2001. </span><span
+
O. &amp; Dachs, G.U., 2001. </span><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Gene directed enzyme/prodrug therapy of
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
cancer: historical appraisal and future prospectives. <i>Journal of cellular
-
lang="EN-US">Gene directed enzyme/prodrug therapy of
+
-
cancer: historical appraisal and future prospectives. <i>Journal
+
-
of cellular
+
physiology</i>, 187(1), 22-36. Available at:
physiology</i>, 187(1), 22-36. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/11241346.</span></p>
http://www.ncbi.nlm.nih.gov/pubmed/11241346.</span></p>
-
<p style="text-indent: 36pt;"><span
+
 
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Huber,
+
<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Huber,
-
B.E. et al., 1993. </span><span
+
B.E. et al., 1993. </span><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">In vivo antitumor activity of 5-fluorocytosine on human
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
colorectal carcinoma cells genetically modified to express cytosine deaminase. <i>Cancer
-
lang="EN-US">In vivo antitumor activity of 5-fluorocytosine
+
-
on human
+
-
colorectal carcinoma cells genetically modified to express cytosine
+
-
deaminase. <i>Cancer
+
research</i>, 53(19), 4619-26. Available at:
research</i>, 53(19), 4619-26. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/8402637.</span></p>
http://www.ncbi.nlm.nih.gov/pubmed/8402637.</span></p>
-
<p style="text-indent: 36pt;"><span
+
 
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Huber,
+
<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Huber,
-
B.E. et al., 1994. </span><span
+
B.E. et al., 1994. </span><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Metabolism of 5-fluorocytosine to 5-fluorouracil in
-
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+
human colorectal tumor cells transduced with the cytosine deaminase gene:
-
lang="EN-US">Metabolism of 5-fluorocytosine to
+
significant antitumor effects when only a small percentage of tumor cells
-
5-fluorouracil in
+
express cytosine deaminase. <i>Proceedings of the National Academy of Sciences
-
human colorectal tumor cells transduced with the cytosine deaminase
+
of the United States of America</i>, 91(17), 8302-6. Available at:
-
gene:
+
-
significant antitumor effects when only a small percentage of tumor
+
-
cells
+
-
express cytosine deaminase. <i>Proceedings of the National
+
-
Academy of Sciences
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of the United States of America</i>, 91(17), 8302-6. Available
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http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=44594&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=44594&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
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lang="EN-US">Moolten, F.L., 1986. Tumor chemosensitivity
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conferred by inserted herpes thymidine kinase genes: paradigm for a prospective
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conferred by inserted herpes thymidine kinase genes: paradigm for a
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cancer control strategy. <i>Cancer research</i>, 46(10), 5276-81. Available at:
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prospective
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cancer control strategy. <i>Cancer research</i>, 46(10),
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5276-81. Available at:
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http://www.ncbi.nlm.nih.gov/pubmed/3019523.</span></p>
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<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Roche, <i>Apoptosis , Cell Death and Cell
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M.E., 2006. A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances
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M.E., 2006. A guanylate kinase/HSV-1 thymidine kinase fusion protein
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prodrug-mediated cell killing. <i>Gene therapy</i>, 13(17), 1309-12. Available
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enhances
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prodrug-mediated cell killing. <i>Gene therapy</i>,
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13(17), 1309-12. Available
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at: http://www.ncbi.nlm.nih.gov/pubmed/16810197.</span></p>
at: http://www.ncbi.nlm.nih.gov/pubmed/16810197.</span></p>
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Revision as of 00:10, 28 October 2010

Contents

Arming: Suicide Genes as GOIs. 1

Introduction. 1

Successful Assembly of Vector Plasmids Carrying Suicide Genes via Cloning. 1

Monitoring Efficient Tumor Killing by Phase-Contrast Microscopy. 4

Quantitative Analysis of Cell Death by Flow Cytometry. 6

Titrating Ganciclovir Concentrations for Efficient Cell Killing by Cytotoxicity Assays. 8

Killing Untransduced Tumor Cells via Bystander Effect 13

Conclusions. 16

References. 16

 

Arming: Suicide Genes as GOIs

Introduction

Gene delivery using viral vectors to specifically target tumor cells gained increasing attention in the last years being efficient in combination with suicide gene approaches (Willmon et al. 2006). Several prodrug/enzyme combinations have been reported. Two systems - ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV-TK) (Ardiani et al. 2010) and 5-fluorocytosine/cytosine deaminase (CD) (Fuchita et al. 2009a) – have been widely used and their therapeutic benefit was demonstrated in preclinical studies (Greco & Dachs 2001). Adeno-associated viruses (AAV) as delivery vectors are commonly used in suicide gene therapy. The suicide gene flanked by the inverted terminal repeats (ITRs) is encapsulated into the virus particles and delivered to the target cells where suicide gene expression is mediated by cellular proteins.

The iGEM team Freiburg_Bioware 2010 provides both the cytosine deaminase (CD, BBa_K404112) and an improved guanylate kinase - thymidine kinase fusion gene (mGMK_TK, BBa_K404113) within the Virus Construction Kit as effective suicide genes. We demonstrate efficient and specific killing of tumor cells by enzymatic cytotoxicity assays, flow cytometry, as well as phase contrast microscopy. HT1080 cancer cell lines were transduced with directed viral particles containing the suicide genes packaged into the viral capsids.

Successful Assembly of Vector Plasmids Carrying Suicide Genes via Cloning

To create the functional vector plasmids, assembly of the constructs carrying the suicide genes was performed following the BioBrick Standard Assembly. All plasmids contain the enhancer-element human beta-globin intron (BBa_K404107) and the human growth hormone terminator signal (hGH, BBa_K404108) flanked by the inverted terminal repeats (ITRs, BBa_K404100 and BBa_K404101). Assembled suicide genes are either under the control of the CMV promoter (BBa_J52034) or the tumor-specific telomerase promoter phTERT (BBa_K404106).

Figure 1: BioBrick-compatible assembly of functional vector plasmids containing the suicide genes. The schematic figure shows the cloning strategy of the guanylate kinase – thymidine kinase fusion gene (mGMK_TK30).

 

To enable modularization of the thymidine kinase mutants TK30 and SR39 (BBa_K404109 and BBa_K404110) according to the BioBrick standard, the fusion genes mGMK_TK30 and mGMK_SR39 (BBa_K404113 and BBa_K404315) and CD (BBa_K404112) were modified using the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) for deletion of iGEM RFC10 pre- and suffix restriction sites. Figure 1 demonstrates one example of successful deletion of a PstI restriction site located within the mGMK_TK30 sequence at position 3109. A point mutation was introduced replacing the nucleotide G by A, resulting in the deletion of the restriction site while maintaining the encoded amino acid glutamine. Exchange of guanine to adenine was confirmed by sequencing (Figure 2).

Figure 2: Cytosine to guanine exchange by site-directed mutagenesis using QuikChange Lightning Kit provided by Stratagene was successful as demonstrated by (A) test digestion linearizing the plasmid with PstI and (B) by sequencing.

 

Furthermore, assembly of BioBrick-compatible vector plasmids was performed. An example for the last assembly step of mGMK_TK30 and hGH_rITR is shown in Figure 3. The plasmids were digested with both XbaI and PstI (New England Biolabs, Insert: BBa_K404116: hGH_rITR) or SpeI and PstI (Vector) and loaded on an agarose gel. As demonstrated in the preparative gel in Figure 3, the expected bands were detected under UV light and the extracted, subsequently purified DNA was successfully ligated and transformed into E. coli. Each assembly step for producing the BioBricks was conducted following the iGEM BioBrick standard.

Figure 3: Assembly of mGMK_TK30 (vector molecule, in pSB1C3) and hGH-terminator_rightITR (insert molecule). The digested fragments were visualized under UV-light and correspond to the expected sizes.

 

Monitoring Efficient Tumor Killing by Phase-Contrast Microscopy

Tumor cells, transduced with viral particles encapsidating the effector constructs containing the mGMK_TK30 driven by the CMV promoter, were cultured both in presence and absence of ganciclovir (Roche). Morphological changes were monitored via phase-contrast microscopy for 48 hours post infection. As it can be seen in Figure 4, non-transduced tumor cells treated with ganciclovir and transduced cells without ganciclovir did not show significant cell ablation. In contrast, transduced cells expressing the guanylate kinase - thymidine kinase fusion protein showed significant cell death after incubation with ganciclovir for 48 hours post infection.

A Beschreibung: http://2010.igem.org/wiki/images/3/3e/HT_negativ_control_ganciclovir_only.jpg

B Beschreibung: http://2010.igem.org/wiki/images/f/f0/HT_TKGMK_clone_1_ohne_Ganciclovir_300%C2%B5l.jpg

C

Beschreibung: http://2010.igem.org/wiki/images/c/c3/HT_TKGMK_clone_1_with_ganciclovir_300%C2%B5l.jpg

D Beschreibung: http://2010.igem.org/wiki/images/7/73/HT_TKGMK_clone_1_with_ganciclovir_600%C2%B5l_well_2.jpg

Figure 4: Qualitative analysis of cell death induced by conversion of ganciclovir to ganciclovir-triphosphate by virus-delivered guanylate - thymidine kinase (mGMK_TK30). A: Non-transduced HT1080 cells incubated in the presence of ganciclovir not exhibiting cell death. B: Untreated transduced HT1080 cells showing a high survival rate. C: HT1080 cells transduced with 300 µL viral particles and incubated with ganciclovir leading to tumor cell ablation. D: HT1080 cells transduced with 600 µL viral particles and incubated with ganciclovir leading to ablation of tumor cells.

 

Suicide gene therapy is based on the localized conversion of non-toxic prodrugs to toxic substances (Greco & Dachs 2001), promoting cell death in the tumor tissue (Figure 5). Directed gene delivery is achieved by using recombinant viral vectors as provided by the iGEM team Freiburg_Bioware 2010 within the Virus Construction Kit.


Figure 5: Overview of the suicide gene therapy approach. Non-toxic prodrugs are converted into toxic effector molecules leading to cell death of the tumor cells.

Non-transduced cells can survive in the presence of ganciclovir since the prodrug is not toxic for these cells (Figure 4A). The demonstration that transduced cells are viable in the absence of ganciclovir confirms that cell killing is indeed induced by combination of the delivered thymidine kinase and treatment with ganciclovir. Viral particles encapsidating the suicide construct mGMK_TK30 are efficient in directed gene delivery, thus leading to cell death of transduced cells due to overexpression of mGMK_TK30 and prodrug conversion. The cell-toxic ganciclovir-triphosphate is incorporated into the nascent DNA chain leading to replication termination and finally resulting in death of dividing cells.

Quantitative Analysis of Cell Death by Flow Cytometry

Quantitative analysis of the cytotoxic effect induced by mGMK_TK30 was first conducted by flow cytometry analysis 72 hours post transduction. HT1080 cells were stained with 7-AAD and Annexin V. 7-AAD intercalates in double-stranded DNA after penetrating cell membranes of dead cells, whereas Annexin V specifically binds phosphatidylserine which is only accessible during apoptosis. Figure 6 demonstrates the relation between cell death and ganciclovir concentration.

 

Figure 6: Flow cytometry data analysis. A: Gating non-transduced HT1080 cells (control). B: Non-transduced cells without staining plotted against 7-AAD. C: Gating non-transduced cells stained with 7-AAD. D: Non-transduced, 7-AAD-stained cells plotted against 7-AAD. E: Gating transduced cells (GOI: mGMK_TK30) treated with 485 µM ganciclovir. F: Gated, Annexin-V stained cells plotted against AnnV-2 Log.  G: Gated cells  plotted against 7-AAD H: Gated, 7-AAD and Annexin-V stained cells plotted against 7-AAD and Annexin-V. Gate R19 comprised Annexin-V and 7-AAD positive cells.

 

 

Figure 7: Quantification of flow cytometry data provided in Figure 6. With increasing ganciclovir concentration, the survival rate of cells decreases. 60 % of HT1080 cells treated with 4.85 mM ganciclovir show tumor ablation, however even lower amounts of ganciclovir lead to significant cell death.

 

The effect of different ganciclovir concentrations on transduced HT1080 sarcoma cells was investigated. Transduction was performed with recombinant viral particles encapsidating the mGMK_TK30 prodrug gene. 72 hours post infection, cells were stained with 7-AAD and Annexin V. As Figure 7 shows, the fraction of killed transduced cells is proportional to the applied ganciclovir concentration.

Titrating Ganciclovir Concentrations for Efficient Cell Killing by Cytotoxicity Assays

Further analysis of the cytotoxic effect induced by thymidine kinase converting ganciclovir to the toxic anti-metabolite has been performed using MTT assays. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide), also known as MTT, is a yellow-colored tetrazole, which is reduced to purple insoluble formazan in the presence of NADH and NADPH (Roche n.d.). Colorimetric analysis can be carried out via spectrometry. Different tumor cell lines, HT1080 and A431, were transduced with the recombinant viruses carrying the linear DNA construct coding for mGMK-TK30 regulated by the CMV promoter and subsequently treated with ganciclovir. 48 and 72 hours post infection, cells were incubated with MTT and fresh DMEM. After cell lysis by DMSO, absorbance of formazan at 570 nm was quantified using a Tecan Sunrise plate reader.

A

B

Figure 8: Effect of ganciclovir on HT1080 cell survival 72 hours post infection as (A) two dimensional plot of survival of cells and (B) three-dimensional plot of ganciclovir, virus particles and cell survival.

 

 

 

A  

B

Figure 9: Effect of ganciclovir on HT1080 cell survival 96 hours post infection. (A) two dimensional plot of cell survival  (B) three-dimensional plot of ganciclovir, virus particles and cell survival.

 

Data of MTT assay quantification are shown in Figure 8 and Figure 9. HT1080 cells were infected with viral particles containing the mGMK_TK30 transgene.  72 h- and 96 h post infection and addition of ganciclovir, cells were incubated with MTT. Changes in absorbance were measured and survival of cells plotted against ganciclovir concentration. Figure 8A demonstrates the correlation between increasing ganciclovir concentrations and percentage of cell survival. Furthermore, different virus particle concentrations were used for transduction. Figure 8B shows that the highest amount of viral particles combined with the highest ganciclovir concentration led to significant HT1080 apoptosis 72 hours post transduction.

Additionally, 96 hours post infection cells were incubated with MTT and absorbance was quantified via spectrometry (Figure 9). Again, survival of HT1080 cells was plotted against increasing ganciclovir concentrations.

Killing Non-transduced Tumor Cells via Bystander Effect

The bystander effect was first reported by Moolten (1986) showing that prodrug convertase negative cells surrounded by suicide enzyme positive cells did not survive prodrug treatment. Besides efficient killing of targeted tumor cells, neighboring, non-transduced cells are killed as well, providing an important effect in cancer treatment. Since 5-Fluorouracil is soluble and can diffuse into adjacent cells (Huber et al. 1993) (Huber et al. 1994), the bystander effect was demonstrated using cytosine deaminase as gene of interest.

 

Figure 10: Schematic overview of the Bystander effect.

 

The aim was to investigate if the modified AAV-2 is able to kill tumor cells with the cytosine deaminase (CD) as gene of interest. The viral particles were produced according to the standard protocol using the following plasmids:

·         pHelper

·         Rep/Cap: pSB1C3_001_[AAV2]-Rep-VP123(ViralBrick-587KO-empty)_p5-TATAless

·         Gene of interest: pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_CD_hGH_[AAV2]-right-ITR

90 % confluent HT1080 cells were transduced in T75 flasks with 9 ml of viral stock. In parallel, another T75 flask was transduced with a viral stock packaged with the mVenus coding sequence to assess transgene expression.

24 hours before harvesting the CD-transduced cells, two six well plates with 200.000 cells per well were seeded. After 30 hours, mVenus expression was observed and the CD-transduced HT1080 were harvested. To minimize the influence of viral particles in the medium, the cells were washed four times with PBS. The cells were counted via a Neubauer cell chamber and 100.000 cells per well of a six well plate were seeded. Additionally, 100.000 of the CD-transduced cells were seeded onto previously seeded untransduced HT1080.

The incubation with the prodrug 5-fluorocytosine (5-FC) was performed at a final concentration of 53 mM. According to Fuchita et al., this amount should be sufficient to show the functionality of the CD (Fuchita et al. 2009b). As demonstrated in Figure 11, cytotoxicity of 5-FC is remarkable.

Figure 11: Transduction of HT1080 with cytosine deaminase-packed viral particles. 5-FC: 5-fluorocytosine (53 mM)

 

After three days of incubation in 5-fluorocytosine, the cells were washed, detached with Trypsin, centrifuged at 200 g for 5 min followed by two washing steps with PBS and finally resuspended with 200 µl DMEM. Living cells were then counted via Trypan blue staining.

After this successful qualitative demonstration of an AAV2-mediated cytosine deaminase treatment, the bystander effect was quantified as well. The activated 5-FC molecules are able to diffuse through the plasma membrane and thus effect cells that are not transduced (Figure 12). The bystander effect was tested with untransduced HT1080 cells, which were mixed with the CD-transduced HT1080 cells.

Figure 12: Quantifying the bystander effect on HT1080 cells  5-FC: 5-fluorocytosine (53 mM)

 

The cytosine deaminase expressing cells have an obvious effect on the viability of the non-transduced cells. As shown in the graph we were able to demonstrate that cytosine deaminase mediated cancer cell death is also fatal for non-transduced cells in close proximity.

Conclusions

Efficient and tissue-specific tumor killing is one major challenge in cancer therapy (Black et al. 1996). Gene-directed enzyme prodrug therapy (GDEPT) is based on the conversion of non-toxic substances into toxic drugs resulting in tumor cell death. The iGEM team Freiburg_Bioware 2010 provides several functional suicide genes within the Virus Construction Kit, thus offering a feasible and modular tool to the growing field of personalized medicine and the iGEM community. We successfully demonstrated cancer cell death caused by the introduction of cytosine deaminase and modified fusion genes consisting of guanylate and thymidine kinases.

 To prevent systemic toxic side effects of conventional chemotherapy, the iGEM team Freiburg_Bioware 2010 took a leap and efficiently retargeted the viral vector for directed suicide gene delivery towards tumor cells. Capsid engineering was successfully demonstrated by the iGEM team Freiburg_Bioware 2010. Further details can be found under Results – Targeting.

References

Ardiani, A., Sanchez-Bonilla, M. & Black, M.E., 2010. Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo. Cancer gene therapy, 17(2), 86-96. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2808426&tool=pmcentrez&rendertype=abstract.

Black, M.E. et al., 1996. Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proceedings of the National Academy of Sciences of the United States of America, 93(8), 3525-9. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=39643&tool=pmcentrez&rendertype=abstract.

Fuchita, M. et al., 2009. Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo. Cancer research, 69(11), 4791-9. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2765227&tool=pmcentrez&rendertype=abstract.

Fuchita, M. et al., 2009. Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo. Cancer research, 69(11), 4791-9. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2765227&tool=pmcentrez&rendertype=abstract.

Greco, O. & Dachs, G.U., 2001. Gene directed enzyme/prodrug therapy of cancer: historical appraisal and future prospectives. Journal of cellular physiology, 187(1), 22-36. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11241346.

Huber, B.E. et al., 1993. In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer research, 53(19), 4619-26. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8402637.

Huber, B.E. et al., 1994. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proceedings of the National Academy of Sciences of the United States of America, 91(17), 8302-6. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=44594&tool=pmcentrez&rendertype=abstract.

Moolten, F.L., 1986. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer research, 46(10), 5276-81. Available at: http://www.ncbi.nlm.nih.gov/pubmed/3019523.

Roche, Apoptosis , Cell Death and Cell Proliferation,

Willmon, C.L., Krabbenhoft, E. & Black, M.E., 2006. A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing. Gene therapy, 13(17), 1309-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16810197.