Team:Freiburg Bioware/Project/Results/Arming

From 2010.igem.org

(Difference between revisions)
Line 2: Line 2:
<html>
<html>
-
<div class=WordSection1>
+
<head>
-
 
+
</head>
-
<p class=MsoTocHeading>Contents</p>
+
<body>
-
 
+
<div class="WordSection1">
-
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc275981841"><span
+
<p class="MsoTocHeading">Contents</p>
-
lang=EN-US>Arming: Suicide Genes as GOIs</span><span style='color:windowtext;
+
<p class="MsoToc2"><span class="MsoHyperlink"><a
-
display:none;text-decoration:none'>. </span><span
+
href="#_Toc275981841"><span lang="EN-US">Arming:
-
style='color:windowtext;display:none;text-decoration:none'>1</span></a></span></p>
+
Suicide Genes as GOIs</span><span
-
 
+
style="color: windowtext; display: none; text-decoration: none;">.
-
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275981842"><span
+
</span><span
-
lang=EN-US>Introduction</span><span style='color:windowtext;display:none;
+
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
-
text-decoration:none'>. </span><span
+
<p class="MsoToc3"><span class="MsoHyperlink"><a
-
style='color:windowtext;display:none;text-decoration:none'>1</span></a></span></p>
+
href="#_Toc275981842"><span lang="EN-US">Introduction</span><span
-
 
+
style="color: windowtext; display: none; text-decoration: none;">.
-
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275981843"><span
+
</span><span
-
lang=EN-US>Successful Assembly of Vector Plasmids Carrying Suicide Genes via
+
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
-
Cloning</span><span style='color:windowtext;display:none;text-decoration:none'>. </span><span
+
<p class="MsoToc3"><span class="MsoHyperlink"><a
-
style='color:windowtext;display:none;text-decoration:none'>1</span></a></span></p>
+
href="#_Toc275981843"><span lang="EN-US">Successful
-
 
+
Assembly of Vector Plasmids Carrying Suicide Genes via
-
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275981844"><span
+
Cloning</span><span
-
lang=EN-US>Monitoring Efficient Tumor Killing by Phase-Contrast Microscopy</span><span
+
style="color: windowtext; display: none; text-decoration: none;">.
-
style='color:windowtext;display:none;text-decoration:none'>. </span><span
+
</span><span
-
style='color:windowtext;display:none;text-decoration:none'>4</span></a></span></p>
+
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
-
 
+
<p class="MsoToc3"><span class="MsoHyperlink"><a
-
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275981845"><span
+
href="#_Toc275981844"><span lang="EN-US">Monitoring
-
lang=EN-US>Quantitative Analysis of Cell Death by Flow Cytometry</span><span
+
Efficient 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;">.
-
style='color:windowtext;display:none;text-decoration:none'>6</span></a></span></p>
+
</span><span
-
 
+
style="color: windowtext; display: none; text-decoration: none;">4</span></a></span></p>
-
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275981846"><span
+
<p class="MsoToc3"><span class="MsoHyperlink"><a
-
lang=EN-US>Titrating Ganciclovir Concentrations for Efficient Cell Killing by
+
href="#_Toc275981845"><span lang="EN-US">Quantitative
-
Cytotoxicity Assays</span><span style='color:windowtext;display:none;
+
Analysis of Cell Death by Flow Cytometry</span><span
-
text-decoration:none'>. </span><span
+
style="color: windowtext; display: none; text-decoration: none;">.
-
style='color:windowtext;display:none;text-decoration:none'>8</span></a></span></p>
+
</span><span
-
 
+
style="color: windowtext; display: none; text-decoration: none;">6</span></a></span></p>
-
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275981847"><span
+
<p class="MsoToc3"><span class="MsoHyperlink"><a
-
lang=EN-US>Killing Untransduced Tumor Cells via Bystander Effect</span><span
+
href="#_Toc275981846"><span lang="EN-US">Titrating
-
style='color:windowtext;display:none;text-decoration:none'> </span><span
+
Ganciclovir Concentrations for Efficient Cell Killing by
-
style='color:windowtext;display:none;text-decoration:none'>13</span></a></span></p>
+
Cytotoxicity Assays</span><span
-
 
+
style="color: windowtext; display: none; text-decoration: none;">.
-
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275981848"><span
+
</span><span
-
lang=EN-US>Conclusions</span><span style='color:windowtext;display:none;
+
style="color: windowtext; display: none; text-decoration: none;">8</span></a></span></p>
-
text-decoration:none'>. </span><span
+
<p class="MsoToc3"><span class="MsoHyperlink"><a
-
style='color:windowtext;display:none;text-decoration:none'>16</span></a></span></p>
+
href="#_Toc275981847"><span lang="EN-US">Killing
-
 
+
Untransduced Tumor Cells via Bystander Effect</span><span
-
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275981849"><span
+
style="color: windowtext; display: none; text-decoration: none;">
-
lang=EN-US>References</span><span style='color:windowtext;display:none;
+
</span><span
-
text-decoration:none'>. </span><span
+
style="color: windowtext; display: none; text-decoration: none;">13</span></a></span></p>
-
style='color:windowtext;display:none;text-decoration:none'>16</span></a></span></p>
+
<p class="MsoToc3"><span class="MsoHyperlink"><a
-
 
+
href="#_Toc275981848"><span lang="EN-US">Conclusions</span><span
-
<p class=MsoNormal>&nbsp;</p>
+
style="color: windowtext; display: none; text-decoration: none;">.
-
 
+
</span><span
-
<h2><a name="_Toc275981841"><span lang=EN-US>Arming: Suicide Genes as GOIs</span></a></h2>
+
style="color: windowtext; display: none; text-decoration: none;">16</span></a></span></p>
-
 
+
<p class="MsoToc3"><span class="MsoHyperlink"><a
-
<h3><a name="_Toc275981842"><span lang=EN-US>Introduction</span></a></h3>
+
href="#_Toc275981849"><span lang="EN-US">References</span><span
-
 
+
style="color: windowtext; display: none; text-decoration: none;">.
-
<p class=MsoNormal><span lang=EN-US>Gene delivery using viral vectors to
+
</span><span
-
specifically target tumor cells gained increasing attention in the last years
+
style="color: windowtext; display: none; text-decoration: none;">16</span></a></span></p>
-
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
+
<p class="MsoNormal">&nbsp;</p>
-
combinations have been reported. The two systems - ganciclovir (GCV)/herpes
+
<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>
 +
<p class="MsoNormal"><span lang="EN-US">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 </span><span
 +
lang="EN-US">(Willmon et al. 2006)</span><span
 +
lang="EN-US">. Several prodrug/enzyme
 +
combinations have been reported. The two systems - ganciclovir
 +
(GCV)/herpes
simplex virus thymidine kinase (HSV-TK) </span><span
simplex virus thymidine kinase (HSV-TK) </span><span
-
lang=EN-US>(Ardiani et al. 2010)</span><span lang=EN-US> and
+
lang="EN-US">(Ardiani et al. 2010)</span><span
 +
lang="EN-US"> and
5-fluorocytosine/cytosine deaminase (CD) </span><span
5-fluorocytosine/cytosine deaminase (CD) </span><span
-
lang=EN-US>(Fuchita et al. 2009a)</span><span lang=EN-US> – have been widely
+
lang="EN-US">(Fuchita et al. 2009a)</span><span
-
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
+
lang="EN-US"> – have been widely
-
viruses (AAV) as delivery vectors are commonly used in suicide gene therapy. The
+
used and their therapeutic benefit was demonstrated in preclinical
-
suicide gene flanked by the inverted terminal repeats (ITRs) is encapsulated
+
studies </span><span lang="EN-US">(Greco &amp;
-
into the virus particles and delivered to the target cells where suicide gene
+
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>
-
 
+
<p class="MsoNormal"><span lang="EN-US">The
-
<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware 2010 provides
+
iGEM team Freiburg_Bioware 2010 provides
-
both the cytosine deaminase (CD, BBa_K404112) and an improved guanylate kinase
+
both the cytosine deaminase (CD, BBa_K404112) and an improved guanylate
 +
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 lines were
+
cytometry, as well as phase contrast microscopy. HT1080 cancer cell
-
transduced with directed viral particles containing the suicide genes packaged
+
lines were
 +
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
-
<h3><a name="_Toc275981843"><span lang=EN-US>Successful Assembly of Vector Plasmids
+
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
-
<p class=MsoNormal><span lang=EN-US>Assembly of the constructs carrying the
+
of the constructs carrying the
-
suicide genes (termed vector plasmids) was performed following the BioBrick
+
suicide genes (termed vector plasmids) was performed following the
 +
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 or the tumor-specific telomerase promoter phTERT (BBa_K404106). </span></p>
+
BBa_K404100 and
-
 
+
BBa_K404101). Assembled suicide genes are either under the control of
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+
the CMV
-
 
+
promoter or the tumor-specific telomerase promoter phTERT
-
<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
+
(BBa_K404106). </span></p>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<div align=center>
+
lang="EN-US">&nbsp;</span></p>
-
 
+
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0
+
<div align="center">
-
  style='border-collapse:collapse;border:none'>
+
<table class="MsoTableGrid"
-
<tr style='height:343.9pt'>
+
  style="border: medium none ; border-collapse: collapse;"
-
  <td width=581 valign=top style='width:435.8pt;padding:0cm 5.4pt 0cm 5.4pt;
+
border="0" cellpadding="0" cellspacing="0">
-
  height:343.9pt'>
+
  <tbody>
-
  <p class=MsoNormal style='text-indent:0cm;page-break-after:avoid'><img
+
    <tr style="height: 343.9pt;">
-
  width=573 height=388 id="Grafik 9"
+
      <td
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image001.png"></p>
+
style="padding: 0cm 5.4pt; width: 435.8pt; height: 343.9pt;"
-
  <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
+
valign="top" width="581">
-
  vector plasmids containing the suicide genes. The schematic figure shows the
+
      <p class="MsoNormal"
-
  cloning strategy of the guanylate kinase – thymidine kinase fusion gene
+
style="text-indent: 0cm; page-break-after: avoid;"><img
-
  (mGMK_TK30).</span></p>
+
style="width: 573px; height: 388px;" alt="" id="Grafik 9"
-
  </td>
+
src="http://2010.igem.org/wiki/images/9/9b/Freiburg10_BBA_overview_suicide.png"></p>
-
</tr>
+
      <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>
</table>
-
 
</div>
</div>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+
lang="EN-US">&nbsp;</span></p>
-
 
+
<p class="MsoNormal"><span lang="EN-US">In
-
<p class=MsoNormal><span lang=EN-US>In order to modularize thymidine kinase
+
order to modularize thymidine kinase
-
mutants TK30 and SR39 (BBa_K404109 and BBa_K404110) according to the BioBrick
+
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 </span><span
standard, the fusion genes mGMK_TK30 and mGMK_SR39 (BBa_K404113 and </span><span
-
lang=EN-US style='line-height:200%;color:black'>BBa_K404315</span><span
+
style="line-height: 200%; color: black;" lang="EN-US">BBa_K404315</span><span
-
lang=EN-US>) and CD (</span><span lang=EN-US style='line-height:200%;
+
lang="EN-US">) and CD (</span><span
-
color:black'>BBa_K404112</span><span lang=EN-US>), were modified using the QuikChange
+
style="line-height: 200%; color: black;" lang="EN-US">BBa_K404112</span><span
-
Lightning Site-Directed Mutagenesis Kit (Stratagene) for deletion of iGEM pre-
+
lang="EN-US">), were modified using the QuikChange
-
and suffix restriction sites. </span><span lang=EN-US>Figure 1</span><span
+
Lightning Site-Directed Mutagenesis Kit (Stratagene) for deletion of
-
lang=EN-US> demonstrates one example of successful deletion of the PstI
+
iGEM pre-
-
restriction site located within the mGMK_TK30 sequence at position 3109. Base
+
and suffix restriction sites. </span><span lang="EN-US">Figure
-
pair exchange was introduced by replacing the nucleotide G with A, resulting in
+
1</span><span lang="EN-US"> demonstrates one example
-
the deletion of the restriction site, but maintaining the amino acid glutamine.
+
of successful deletion of the PstI
-
Successful transition of G to A was confirmed by sequencing (</span><span lang=EN-US>Figure 2</span><span lang=EN-US>). </span></p>
+
restriction site located within the mGMK_TK30 sequence at position
-
 
+
3109. Base
-
<div align=center>
+
pair exchange was introduced by replacing the nucleotide G with A,
-
 
+
resulting in
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 width=639
+
the deletion of the restriction site, but maintaining the amino acid
-
  style='width:479.55pt;border-collapse:collapse;border:none'>
+
glutamine.
-
  <tr style='height:179.55pt'>
+
Successful transition of G to A was confirmed by sequencing (</span><span
-
  <td width=639 valign=top style='width:479.55pt;padding:0cm 5.4pt 0cm 5.4pt;
+
lang="EN-US">Figure 2</span><span lang="EN-US">).
-
  height:179.55pt'>
+
</span></p>
-
  <p class=MsoNormal style='text-indent:0cm;page-break-after:avoid'><img
+
<div align="center">
-
  width=635 height=328 id="Grafik 81"
+
<table class="MsoTableGrid"
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image002.png"></p>
+
  style="border: medium none ; width: 479.55pt; border-collapse: collapse;"
-
  <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
+
border="0" cellpadding="0" cellspacing="0"
-
  site-directed mutagenesis using QuikChange Lightning Kit provided by
+
  width="639">
-
  Stratagene has been successful performed as demonstrated by (A) test
+
  <tbody>
-
  digestion linearizing the plasmid with PstI and (B) by sequencing.</span></p>
+
    <tr style="height: 179.55pt;">
-
  </td>
+
      <td
-
</tr>
+
style="padding: 0cm 5.4pt; width: 479.55pt; height: 179.55pt;"
 +
valign="top" width="639">
 +
      <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>
 +
      <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>
</table>
-
 
</div>
</div>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+
lang="EN-US">&nbsp;</span></p>
-
 
+
<p class="MsoNormal"><span lang="EN-US">Furthermore,
-
<p class=MsoNormal><span lang=EN-US>Furthermore, assembly of BioBrick-compatible
+
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
+
mGMK_TK30 and hGH_rITR is shown in </span><span lang="EN-US">Figure
-
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 (Insert: BBa_K404116: hGH_rITR) or SpeI and PstI (Vector) and
+
digested with
-
loaded on an agarose gel. As demonstrated in the preparative gel in </span><span lang=EN-US>Figure 3</span><span lang=EN-US>, the expected bands were detected
+
both XbaI and PstI (Insert: BBa_K404116: hGH_rITR) or SpeI and PstI
-
under UV light and the extracted DNA was be successfully ligated. Each assembly
+
(Vector) and
 +
loaded on an agarose gel. As demonstrated in the preparative gel in </span><span
 +
lang="EN-US">Figure 3</span><span lang="EN-US">,
 +
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
step for producing BioBricks was conducted following the iGEM BioBrick
standard.</span></p>
standard.</span></p>
-
 
+
<div align="center">
-
<div align=center>
+
<table class="MsoTableGrid"
-
 
+
  style="border: medium none ; width: 387.35pt; border-collapse: collapse;"
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 width=516
+
border="0" cellpadding="0" cellspacing="0"
-
  style='width:387.35pt;border-collapse:collapse;border:none'>
+
  width="516">
-
  <tr style='height:125.95pt'>
+
  <tbody>
-
  <td width=516 valign=top style='width:387.35pt;padding:0cm 5.4pt 0cm 5.4pt;
+
    <tr style="height: 125.95pt;">
-
  height:125.95pt'>
+
      <td
-
  <p class=MsoNormal style='text-indent:0cm;page-break-after:avoid'><img
+
style="padding: 0cm 5.4pt; width: 387.35pt; height: 125.95pt;"
-
  width=519 height=189 id="Grafik 28"
+
valign="top" width="516">
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image003.png"></p>
+
      <p class="MsoNormal"
-
  <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
+
style="text-indent: 0cm; page-break-after: avoid;"><img
-
  to hGH-terminator_rightITR (insert). The digested fragments correspond to the
+
style="width: 519px; height: 189px;" alt=""
-
  expected sizes.</span></p>
+
id="Grafik 28"
-
  </td>
+
src="http://2010.igem.org/wiki/images/f/f2/Freiburg10_Cloning_mGMK_TK30_Arming.png"></p>
-
</tr>
+
      <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>
</table>
-
 
</div>
</div>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+
lang="EN-US">&nbsp;</span></p>
-
 
+
<h3><a name="_Toc275981844"><span lang="EN-US">Monitoring
-
<h3><a name="_Toc275981844"><span lang=EN-US>Monitoring Efficient Tumor Killing
+
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
-
<p class=MsoNormal><span lang=EN-US>Tumor cells, transduced with viral
+
cells, transduced with viral
-
particles encapsidating the effector constructs containing the mGMK_TK30 driven
+
particles encapsidating the effector constructs containing the
-
by the CMV promoter, were cultured in presence and absence of ganciclovir.
+
mGMK_TK30 driven
-
Morphological changes were monitored via phase-contrast microscopy until 48
+
by the CMV promoter, were cultured in presence and absence of
 +
ganciclovir.
 +
Morphological changes were monitored via phase-contrast microscopy
 +
until 48
hours post infection. As it can be seen in </span><span
hours post infection. As it can be seen in </span><span
-
lang=EN-US>Figure 4</span><span lang=EN-US> non-transduced cells treated with ganciclovir
+
lang="EN-US">Figure 4</span><span lang="EN-US">
-
and transduced cell without ganciclovir did not show significant tumor cell
+
non-transduced cells treated with ganciclovir
-
ablation. In contrast transduced cells expressing the guanylate kinase -
+
and transduced cell without ganciclovir did not show significant tumor
-
thymidine kinase fusion protein, showed significant cell death after incubation
+
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. </span></p>
with ganciclovir for 48 hours post infection. </span></p>
-
 
+
<table class="MsoTableGrid"
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 width=655
+
  style="border: medium none ; width: 491pt; border-collapse: collapse;"
-
  style='width:491.0pt;border-collapse:collapse;border:none'>
+
border="0" cellpadding="0" cellspacing="0"
-
  <tr style='height:188.8pt'>
+
  width="655">
-
  <td width=330 style='width:247.35pt;padding:0cm 5.4pt 0cm 5.4pt;height:188.8pt'>
+
  <tbody>
-
  <p class=MsoNoSpacing><b><span lang=EN-US>A</span></b><span lang=EN-US> </span><img
+
    <tr style="height: 188.8pt;">
-
  width=318 height=238 id="Grafik 1"
+
      <td
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image004.jpg"
+
style="padding: 0cm 5.4pt; width: 247.35pt; height: 188.8pt;"
-
  alt="Beschreibung: http://2010.igem.org/wiki/images/3/3e/HT_negativ_control_ganciclovir_only.jpg"></p>
+
width="330">
-
  </td>
+
      <p class="MsoNoSpacing"><b><span
-
  <td width=325 style='width:243.65pt;padding:0cm 5.4pt 0cm 5.4pt;height:188.8pt'>
+
lang="EN-US">A</span></b><span lang="EN-US">
-
  <p class=MsoNoSpacing><b><span lang=EN-US>B</span></b><span lang=EN-US> </span><img
+
      </span><img style="width: 318px; height: 238px;"
-
  width=318 height=238 id="Grafik 2"
+
id="Grafik 1"
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image005.jpg"
+
src="http://2010.igem.org/wiki/images/4/4f/Freiburg10_Microscopy_Arming_A.png"
-
  alt="Beschreibung: http://2010.igem.org/wiki/images/f/f0/HT_TKGMK_clone_1_ohne_Ganciclovir_300%C2%B5l.jpg"></p>
+
alt="Beschreibung: http://2010.igem.org/wiki/images/3/3e/HT_negativ_control_ganciclovir_only.jpg"></p>
-
  </td>
+
      </td>
-
</tr>
+
      <td
-
<tr style='height:188.8pt'>
+
style="padding: 0cm 5.4pt; width: 243.65pt; height: 188.8pt;"
-
  <td width=330 style='width:247.35pt;padding:0cm 5.4pt 0cm 5.4pt;height:188.8pt'>
+
width="325">
-
  <p class=MsoNoSpacing><b><span lang=EN-US>C</span></b><span lang=EN-US> </span></p>
+
      <p class="MsoNoSpacing"><b><span
-
  <p class=MsoNoSpacing style='page-break-after:avoid'><img width=318
+
lang="EN-US">B</span></b><span lang="EN-US">
-
  height=238 id="Grafik 3"
+
      </span><img style="width: 318px; height: 238px;"
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image006.jpg"
+
id="Grafik 2"
-
  alt="Beschreibung: http://2010.igem.org/wiki/images/c/c3/HT_TKGMK_clone_1_with_ganciclovir_300%C2%B5l.jpg"></p>
+
src="http://2010.igem.org/wiki/images/e/e8/Freiburg10_Microscopy_Arming_B.png"
-
  </td>
+
alt="Beschreibung: http://2010.igem.org/wiki/images/f/f0/HT_TKGMK_clone_1_ohne_Ganciclovir_300%C2%B5l.jpg"></p>
-
  <td width=325 style='width:243.65pt;padding:0cm 5.4pt 0cm 5.4pt;height:188.8pt'>
+
      </td>
-
  <p class=MsoNoSpacing><b><span lang=EN-US>D</span></b><span lang=EN-US> </span><img
+
    </tr>
-
  width=318 height=238 id="Grafik 4"
+
    <tr style="height: 188.8pt;">
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image007.jpg"
+
      <td
-
  alt="Beschreibung: http://2010.igem.org/wiki/images/7/73/HT_TKGMK_clone_1_with_ganciclovir_600%C2%B5l_well_2.jpg"></p>
+
style="padding: 0cm 5.4pt; width: 247.35pt; height: 188.8pt;"
-
  </td>
+
width="330">
-
</tr>
+
      <p class="MsoNoSpacing"><b><span
-
<tr style='height:34.2pt'>
+
lang="EN-US">C</span></b><span lang="EN-US">
-
  <td width=655 colspan=2 style='width:491.0pt;padding:0cm 5.4pt 0cm 5.4pt;
+
      </span></p>
-
  height:34.2pt'>
+
      <p class="MsoNoSpacing" style="page-break-after: avoid;"><img
-
  <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
+
style="width: 318px; height: 238px;" id="Grafik 3"
-
  by conversion of ganciclovir to ganciclovir-triphosphate by virus-delivered
+
src="http://2010.igem.org/wiki/images/a/ac/Freiburg10_Microscopy_Arming_C.png"
-
  guanylate - thymidine kinase (mGMK_TK30). A: Non-transduced HT1080 cells
+
alt="Beschreibung: http://2010.igem.org/wiki/images/c/c3/HT_TKGMK_clone_1_with_ganciclovir_300%C2%B5l.jpg"></p>
-
  incubated in the presence of ganciclovir did not exhibit cell death. B:
+
      </td>
-
  Transduced HT1080 cells untreated resulting in survival of most cells. C:
+
      <td
-
  HT1080 cells were transduced with 300µL viral particles and incubated with
+
style="padding: 0cm 5.4pt; width: 243.65pt; height: 188.8pt;"
-
  ganciclovir leading to ablation of tumor cells. D: HT1080 cells were
+
width="325">
-
  transduced with 600µL viral particles and incubated with ganciclovir leading
+
      <p class="MsoNoSpacing"><b><span
-
  to ablation of tumor cells. </span></p>
+
lang="EN-US">D</span></b><span lang="EN-US">
-
  </td>
+
      </span><img style="width: 318px; height: 238px;"
-
</tr>
+
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>
</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
+
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>, leading to cell
+
lang="EN-US">(Greco &amp; Dachs 2001)</span><span
-
death (</span><span lang=EN-US>Figure 5</span><span lang=EN-US>). Directed gene
+
lang="EN-US">, leading to cell
-
delivery is achieved by using recombinant viral vectors as provided by the iGEM
+
death (</span><span lang="EN-US">Figure 5</span><span
 +
lang="EN-US">). Directed gene
 +
delivery is achieved by using recombinant viral vectors as provided by
 +
the iGEM
team Freiburg_Bioware 2010 within the Virus Construction Kit.</span></p>
team Freiburg_Bioware 2010 within the Virus Construction Kit.</span></p>
-
 
+
<table class="MsoTableGrid"
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left
+
  style="border: medium none ; border-collapse: collapse; margin-left: 4.8pt; margin-right: 4.8pt;"
-
  style='border-collapse:collapse;border:none;margin-left:4.8pt;margin-right:
+
  align="left" border="0" cellpadding="0"
-
4.8pt'>
+
cellspacing="0">
-
  <tr style='height:174.85pt'>
+
  <tbody>
-
  <td width=597 valign=top style='width:447.8pt;padding:0cm 5.4pt 0cm 5.4pt;
+
    <tr style="height: 174.85pt;">
-
  height:174.85pt'>
+
      <td
-
  <p class=MsoNormal style='text-indent:0cm;page-break-after:avoid'><span
+
style="padding: 0cm 5.4pt; width: 447.8pt; height: 174.85pt;"
-
  style='position:relative;z-index:251658240;left:-1px;top:0px;width:583px;
+
valign="top" width="597">
-
  height:271px'><img width=583 height=271
+
      <p class="MsoNormal"
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image008.png"></span><br
+
style="text-indent: 0cm; page-break-after: avoid;"><span
-
  clear=ALL>
+
style="position: relative; z-index: 251658240; left: -1px; top: 0px; width: 583px; height: 271px;"><img
-
  </p>
+
style="width: 583px; height: 271px;" alt=""
-
  <p class=MsoCaption><a name="_Ref275977093"><span lang=EN-US>Figure </span></a><span lang=EN-US>5</span><span lang=EN-US>: Overview over the suicide gene therapy
+
src="http://2010.igem.org/wiki/images/a/a4/Freiburg10_overview_GDEPT.png"></span><br
-
  approach. Non-toxic prodrugs are converted into toxic effector molecules
+
clear="all">
-
  leading to cell death of the tumor cells. </span></p>
+
      </p>
-
  </td>
+
      <p class="MsoCaption"><a name="_Ref275977093"><span
-
</tr>
+
lang="EN-US">Figure </span></a><span
 +
lang="EN-US">5</span><span lang="EN-US">:
 +
Overview over the suicide gene therapy
 +
approach. Non-toxic prodrugs are converted into toxic effector
 +
molecules
 +
leading to cell death of the tumor cells. </span></p>
 +
      </td>
 +
    </tr>
 +
  </tbody>
</table>
</table>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+
lang="EN-US">&nbsp;</span></p>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+
lang="EN-US">&nbsp;</span></p>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>Since ganciclovir
+
lang="EN-US">Since ganciclovir
-
is not toxic for cells, non-transduced cells can survive in the presence of the
+
is not toxic for cells, non-transduced cells can survive in the
-
prodrug (</span><span lang=EN-US>Figure 4</span><span lang=EN-US>A).
+
presence of the
-
Demonstrating that transduced cells are viable in absence of ganciclovir,
+
prodrug (</span><span lang="EN-US">Figure 4</span><span
-
confirms that cell killing is induced by combination of delivered thymidine
+
lang="EN-US">A).
-
kinase and treatment with ganciclovir. Viral particles encapsidating the
+
Demonstrating that transduced cells are viable in absence of
-
suicide construct mGMK_TK30 are efficient in directed gene delivery, thus
+
ganciclovir,
-
leading to cell death of transduced cells due to overexpression of mGMK_TK30
+
confirms that cell killing is induced by combination of delivered
-
and prodrug conversion. The cell toxic ganciclovir-triphosphate is incorporated
+
thymidine
-
into the nascent DNA chain leading to replication termination and finally
+
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>
resulting in death of dividing cells. </span></p>
-
 
+
<h3><a name="_Toc275981845"><span lang="EN-US">Quantitative
-
<h3><a name="_Toc275981845"><span lang=EN-US>Quantitative Analysis of Cell
+
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
-
<p class=MsoNormal><span lang=EN-US>Quantitative analysis of the cytotoxic
+
analysis of the cytotoxic
-
effect induced by mGMK_TK30 was first conducted by flow cytometry analysis 72 hours
+
effect induced by mGMK_TK30 was first conducted by flow cytometry
-
post transduction. HT1080 cells were stained with 7-AAD and Annexin V. 7-AAD
+
analysis 72 hours
-
intercalates in double-stranded DNA after penetrating cell membranes of dead
+
post transduction. HT1080 cells were stained with 7-AAD and Annexin V.
-
cells, whereas Annexin V binds specifically phosphatidylserine which is only
+
7-AAD
-
accessible during apoptosis. </span><span lang=EN-US>Figure 6</span><span
+
intercalates in double-stranded DNA after penetrating cell membranes of
-
lang=EN-US> demonstrates the relation between cell death and ganciclovir
+
dead
 +
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>
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 border=0 cellspacing=0 cellpadding=0 width=637
+
border="0" cellpadding="0" cellspacing="0"
-
  style='width:477.6pt;border-collapse:collapse;border:none'>
+
  width="637">
-
  <tr style='height:248.5pt'>
+
  <tbody>
-
  <td width=322 valign=top style='width:241.8pt;padding:0cm 5.4pt 0cm 5.4pt;
+
    <tr style="height: 248.5pt;">
-
  height:248.5pt'>
+
      <td
-
  <p class=MsoNormal style='text-indent:0cm;page-break-after:avoid'><img
+
style="padding: 0cm 5.4pt; width: 241.8pt; height: 248.5pt;"
-
  width=308 height=326 id="Grafik 24"
+
valign="top" width="322">
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image009.png"></p>
+
      <p class="MsoNormal"
-
  </td>
+
style="text-indent: 0cm; page-break-after: avoid;"><img
-
  <td width=314 valign=top style='width:235.8pt;padding:0cm 5.4pt 0cm 5.4pt;
+
style="width: 308px; height: 326px;" alt=""
-
  height:248.5pt'>
+
id="Grafik 24"
-
  <p class=MsoNormal style='text-indent:0cm;page-break-after:avoid'><img
+
src="http://2010.igem.org/wiki/images/9/9d/Freiburg10_FACS_A_D.png"></p>
-
  width=300 height=326 id="Grafik 36"
+
      </td>
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image010.png"></p>
+
      <td
-
  <p class=MsoCaption>&nbsp;</p>
+
style="padding: 0cm 5.4pt; width: 235.8pt; height: 248.5pt;"
-
  </td>
+
valign="top" width="314">
-
</tr>
+
      <p class="MsoNormal"
-
<tr style='height:38.0pt'>
+
style="text-indent: 0cm; page-break-after: avoid;"><img
-
  <td width=637 colspan=2 valign=top style='width:477.6pt;padding:0cm 5.4pt 0cm 5.4pt;
+
style="width: 300px; height: 326px;" alt=""
-
  height:38.0pt'>
+
id="Grafik 36"
-
  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275977177"><span
+
src="http://2010.igem.org/wiki/images/0/0b/Freiburg10_FACS_E_H.png"></p>
-
  lang=EN-US>Figure </span></a><span lang=EN-US>6</span><span lang=EN-US>: </span><span
+
      <p class="MsoCaption">&nbsp;</p>
-
  lang=EN-US>A: Gating non-transduced HT1080 cells (control). B: Non-transduced
+
      </td>
-
  cells without staining plotted against 7-AAD. C: Gating non-transduced cells
+
    </tr>
-
  stained with 7-AAD. D: Non-transduced, 7-AAD-stained cells plotted against
+
    <tr style="height: 38pt;">
-
  7-AAD. E: Gating transduced cells (GOI: mGMK_TK30) treated with 485µM
+
      <td colspan="2"
-
  Ganciclovir. F: Gated, Annexin-V stained cells plotted against AnnV-2 Log.  G:
+
style="padding: 0cm 5.4pt; width: 477.6pt; height: 38pt;"
-
  Gated cells  plotted against 7-AAD H: Gated, 7-AAD and Annexin-V stained
+
valign="top" width="637">
-
  cells plotted against 7-AAD and Annexin-V. Gate R19 comprised Annexin-V and
+
      <p class="MsoCaption" style="text-indent: 0cm;"><a
-
  7-AAD positive cells.</span></p>
+
name="_Ref275977177"><span lang="EN-US">Figure </span></a><span
-
  </td>
+
lang="EN-US">6</span><span lang="EN-US">:
-
</tr>
+
      </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>
</table>
-
 
</div>
</div>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+
lang="EN-US">&nbsp;</span></p>
-
 
+
<p class="MsoNormal"
-
<p class=MsoNormal align=center style='text-align:center;text-indent:0cm'><span
+
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 border=0 cellspacing=0 cellpadding=0
+
border="0" cellpadding="0" cellspacing="0">
-
  style='border-collapse:collapse;border:none'>
+
  <tbody>
-
<tr>
+
    <tr>
-
  <td width=614 valign=top style='width:460.6pt;padding:0cm 5.4pt 0cm 5.4pt'>
+
      <td style="padding: 0cm 5.4pt; width: 460.6pt;"
-
  <p class=MsoNormal align=center style='text-align:center;text-indent:0cm;
+
valign="top" width="614">
-
  page-break-after:avoid'><img width=562 height=387 id="Diagramm 8"
+
      <p class="MsoNormal"
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image011.png"></p>
+
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
-
  <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
+
align="center"><img style="width: 562px; height: 387px;"
-
  provided in </span><span lang=EN-US>Figure 6</span><span lang=EN-US>. With
+
alt="" id="Diagramm 8"
-
  increasing ganciclovir concentration, the survival rate of cells decreases. 60%
+
src="http://2010.igem.org/wiki/images/2/29/Freiburg10_Diagram_mGMK_effectFACS.png"></p>
-
  of HT1080 cells treated with 4,85 mM ganciclovir show tumor ablation, however
+
      <p class="MsoCaption"><a name="_Ref275977227"><span
-
  even lower amounts of ganciclovir led to significant cell death.</span></p>
+
lang="EN-US">Figure </span></a><span
-
  </td>
+
lang="EN-US">7</span><span lang="EN-US">:
-
</tr>
+
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>
</table>
-
 
</div>
</div>
-
 
+
<p class="MsoNormal"
-
<p class=MsoNormal align=center style='text-align:center;text-indent:0cm'><span
+
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>Effect of different ganciclovir
+
of different ganciclovir
-
concentrations on transduced HT1080 sarcoma cells. Transduction has been
+
concentrations on transduced HT1080 sarcoma cells. Transduction has
-
performed with recombinant viral particles encapsidating the mGMK_TK30 prodrug
+
been
-
gene. 72 hours post infection cells were stained with 7-AAD and Annexin V. As </span><span lang=EN-US>Figure 7</span><span lang=EN-US> shows, the higher the ganciclovir concentration,
+
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 </span><span lang="EN-US">Figure 7</span><span
 +
lang="EN-US"> shows, the higher the ganciclovir
 +
concentration,
the more transduced cells were killed.</span></p>
the more transduced cells were killed.</span></p>
-
 
+
<h3><a name="_Toc275981846"><span lang="EN-US">Titrating
-
<h3><a name="_Toc275981846"><span lang=EN-US>Titrating Ganciclovir
+
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
-
<p class=MsoNormal><span lang=EN-US>Further analysis of the cytotoxic effect
+
analysis of the cytotoxic effect
-
induced by thymidine kinase converting ganciclovir to the toxic anti-metabolite
+
induced by thymidine kinase converting ganciclovir to the toxic
-
has been performed using MTT assays. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium
+
anti-metabolite
-
bromide), also known as MTT, is a yellow tetrazole, which is reduced to purple insoluble
+
has been performed using MTT assays.
 +
3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium
 +
bromide), also known as MTT, is a yellow tetrazole, which is reduced to
 +
purple insoluble
formazan in the presence of NADH and NADPH </span><span
formazan in the presence of NADH and NADPH </span><span
-
lang=EN-US>(Roche n.d.)</span><span lang=EN-US>. The colorimetric analysis can be
+
lang="EN-US">(Roche n.d.)</span><span lang="EN-US">.
-
carried out via spectrometry. Different tumor cell lines, HT1080 and A431, were
+
The colorimetric analysis can be
-
transduced with the recombinant viruses carrying the linear DNA construct
+
carried out via spectrometry. Different tumor cell lines, HT1080 and
-
coding for mGMK-TK30 regulated by the CMV promoter and treated with ganciclovir.
+
A431, were
-
48 and 72 hours post infection cells were incubated with MTT and absorbance of
+
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>
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 border=0 cellspacing=0 cellpadding=0 width=601
+
border="0" cellpadding="0" cellspacing="0"
-
  style='width:451.05pt;border-collapse:collapse;border:none'>
+
  width="601">
-
  <tr style='height:176.7pt'>
+
  <tbody>
-
  <td width=601 valign=top style='width:451.05pt;padding:0cm 5.4pt 0cm 5.4pt;
+
    <tr style="height: 176.7pt;">
-
  height:176.7pt'>
+
      <td
-
  <p class=MsoNormal style='text-indent:0cm'><b><span lang=EN-US>A</span></b><img
+
style="padding: 0cm 5.4pt; width: 451.05pt; height: 176.7pt;"
-
  width=588 height=413 id="Diagramm 95"
+
valign="top" width="601">
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image012.png"></p>
+
      <p class="MsoNormal" style="text-indent: 0cm;"><b><span
-
  </td>
+
lang="EN-US">A</span></b><img
-
</tr>
+
style="width: 588px; height: 413px;" alt=""
-
<tr style='height:176.7pt'>
+
id="Diagramm 95"
-
  <td width=601 valign=top style='width:451.05pt;padding:0cm 3.5pt 0cm 3.5pt;
+
src="http://2010.igem.org/wiki/images/8/86/Freiburg10_MTT_72h_2d.png"></p>
-
  height:176.7pt'>
+
      </td>
-
  <p class=MsoNormal style='text-indent:0cm'><b>B</b> </p>
+
    </tr>
-
  <p class=MsoNormal align=center style='text-align:center;text-indent:0cm;
+
    <tr style="height: 176.7pt;">
-
  page-break-after:avoid'><img width=540 height=372 id="Diagramm 10"
+
      <td
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image013.png"></p>
+
style="padding: 0cm 3.5pt; width: 451.05pt; height: 176.7pt;"
-
  <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
+
valign="top" width="601">
-
  killing 72 hours post infection as (A) two dimensional plot of survival of
+
      <p class="MsoNormal" style="text-indent: 0cm;"><b>B</b>
-
  cells and (B) three-dimensional plot of ganciclovir, virus particles and cell
+
      </p>
-
  survival.</span></p>
+
      <p class="MsoNormal"
-
  </td>
+
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
-
</tr>
+
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>
</table>
-
 
</div>
</div>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US style='font-size:
+
style="font-size: 8pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
-
8.0pt;line-height:200%'>&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 lang=EN-US style='font-size:
+
<div align="center">
-
8.0pt;line-height:200%'>&nbsp;</span></p>
+
<table class="MsoTableGrid"
-
 
+
  style="border: medium none ; width: 415pt; border-collapse: collapse;"
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US style='font-size:
+
border="0" cellpadding="0" cellspacing="0"
-
8.0pt;line-height:200%'>&nbsp;</span></p>
+
  width="553">
-
 
+
  <tbody>
-
<div align=center>
+
    <tr style="height: 176.7pt;">
-
 
+
      <td style="padding: 0cm 5.4pt; width: 415pt; height: 176.7pt;"
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 width=553
+
valign="top" width="553">
-
  style='width:415.0pt;border-collapse:collapse;border:none'>
+
      <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 width=553 valign=top style='width:415.0pt;padding:0cm 5.4pt 0cm 5.4pt;
+
      </span></p>
-
  height:176.7pt'>
+
      <p class="MsoNormal"
-
  <p class=MsoNormal style='text-indent:0cm'><b><span lang=EN-US>A </span></b><span
+
style="text-align: center; text-indent: 0cm;" align="center"><img
-
  lang=EN-US> </span></p>
+
style="width: 607px; height: 427px;" alt=""
-
  <p class=MsoNormal align=center style='text-align:center;text-indent:0cm'><img
+
id="Diagramm 1154"
-
  width=607 height=427 id="Diagramm 1154"
+
src="http://2010.igem.org/wiki/images/2/2a/Freiburg10_MTT_96h_2d.png"></p>
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image014.png"></p>
+
      </td>
-
  </td>
+
    </tr>
-
</tr>
+
    <tr style="height: 176.7pt;">
-
<tr style='height:176.7pt'>
+
      <td style="padding: 0cm 5.4pt; width: 415pt; height: 176.7pt;"
-
  <td width=553 valign=top style='width:415.0pt;padding:0cm 5.4pt 0cm 5.4pt;
+
valign="top" width="553">
-
  height:176.7pt'>
+
      <p class="MsoNormal" style="text-indent: 0cm;"><b>B</b></p>
-
  <p class=MsoNormal style='text-indent:0cm'><b>B</b></p>
+
      <p class="MsoNormal"
-
  <p class=MsoNormal align=center style='text-align:center;text-indent:0cm;
+
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
-
  page-break-after:avoid'><img width=606 height=480 id="Diagramm 1156"
+
align="center"><img style="width: 606px; height: 480px;"
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image015.png"></p>
+
alt="" id="Diagramm 1156"
-
  <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
+
src="http://2010.igem.org/wiki/images/0/0a/Freiburg10_MTT_96h_3d.png"></p>
-
  killing 96 hours post infection as (A) two dimensional plot of survival of
+
      <p class="MsoCaption"><a name="_Ref275977314"><span
-
  cells and (B) three-dimensional plot of ganciclovir, virus particles and cell
+
lang="EN-US">Figure </span></a><span
-
  survival.</span></p>
+
lang="EN-US">9</span><span lang="EN-US">:
-
  </td>
+
Effect of ganciclovir on HT1080 cell
-
</tr>
+
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>
</table>
-
 
</div>
</div>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US style='font-size:
+
style="font-size: 8pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
-
8.0pt;line-height:200%'>&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.  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
-
<p class=MsoNormal><span lang=EN-US>Additionally 96 hours post infection cells
+
96 hours post infection cells
-
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
+
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
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
-
<h3><a name="_Toc275981847"><span lang=EN-US>Killing Untransduced Tumor Cells
+
Untransduced Tumor Cells
-
via Bystander Effect</span></a><span lang=EN-US> </span></h3>
+
via Bystander Effect</span></a><span lang="EN-US">
-
 
+
</span></h3>
-
<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
+
<p class="MsoNormal"><span lang="EN-US">The
-
convertase negative cells surrounded by suicide enzyme positive cells did not
+
bystander effect was first reported by </span><span
-
survive prodrug treatment. Besides efficient killing of targeted tumor cells,
+
lang="EN-US">Moolten (1986)</span><span lang="EN-US">
-
neighboring, non-transduced cells are killed as well, providing an important
+
showing that prodrug
-
effect in treating cancer. Since 5-Fluorouracil is soluble and can diffuse into
+
convertase negative cells surrounded by suicide enzyme positive cells
-
adjacent cells </span><span
+
did not
-
lang=EN-US>(Huber et al. 1993)</span><span lang=EN-US> </span><span
+
survive prodrug treatment. Besides efficient killing of targeted tumor
-
lang=EN-US>(Huber et al. 1994)</span><span lang=EN-US>, the bystander effect
+
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">
-
 
+
<table class="MsoTableGrid"
-
<div align=center>
+
  style="border: medium none ; border-collapse: collapse;"
-
 
+
border="0" cellpadding="0" cellspacing="0">
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0
+
  <tbody>
-
  style='border-collapse:collapse;border:none'>
+
    <tr>
-
<tr>
+
      <td style="padding: 0cm 5.4pt; width: 460.6pt;"
-
  <td width=614 valign=top style='width:460.6pt;padding:0cm 5.4pt 0cm 5.4pt'>
+
valign="top" width="614">
-
  <p class=MsoNormal style='text-indent:0cm;page-break-after:avoid'><img
+
      <p class="MsoNormal"
-
  width=605 height=262 id="Grafik 82"
+
style="text-indent: 0cm; page-break-after: avoid;"><img
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image016.png"></p>
+
style="width: 605px; height: 262px;" alt=""
-
  <p class=MsoCaption><span lang=EN-US>Figure </span><span
+
id="Grafik 82"
-
  lang=EN-US>10</span><span lang=EN-US>: Schematic overview of the Bystander
+
src="http://2010.igem.org/wiki/images/c/c0/Freiburg10_Bystander_effect.png"></p>
-
  effect.</span></p>
+
      <p class="MsoCaption"><span lang="EN-US">Figure
-
  </td>
+
      </span><span lang="EN-US">10</span><span
-
</tr>
+
lang="EN-US">: Schematic overview of the Bystander
 +
effect.</span></p>
 +
      </td>
 +
    </tr>
 +
  </tbody>
</table>
</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"
-
<p class=MsoListParagraphCxSpFirst style='margin-left:53.85pt;text-indent:-18.0pt'><span
+
style="margin-left: 53.85pt; text-indent: -18pt;"><span
-
style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&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"
-
<p class=MsoListParagraphCxSpMiddle style='margin-left:53.85pt;text-indent:
+
style="margin-left: 53.85pt; text-indent: -18pt;"><span
-
-18.0pt'><span lang=EN-US style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
style="font-family: Symbol;" lang="EN-US">·<span
-
</span></span><span lang=EN-US>Rep/Cap:
+
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:
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"
-
<p class=MsoListParagraphCxSpLast style='margin-left:53.85pt;text-indent:-18.0pt'><span
+
style="margin-left: 53.85pt; text-indent: -18pt;"><span
-
lang=EN-US style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&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 %
-
<p class=MsoNormal><span lang=EN-US>90 % confluent HT1080 cells were transduced
+
confluent HT1080 cells were transduced
-
in T75 flasks with 9 ml of viral stock. In parallel, another T75 flask was
+
in T75 flasks with 9 ml of viral stock. In parallel, another T75 flask
 +
was
transduced with a viral stock packaged with mVenus to assess transgene
transduced with a viral stock packaged with mVenus to 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
-
<p class=MsoNormal><span lang=EN-US>The incubation with the prodrug
+
incubation with the prodrug
-
5-fluorocytosine (5-FC) was performed at a final concentration of 53 mM.
+
5-fluorocytosine (5-FC) was performed at a final concentration of 53
-
According to Fuchita <i>et al.,</i> this amount should be sufficient to show
+
mM.
-
the functionality of the CD </span><span
+
According to Fuchita <i>et al.,</i> this amount should be
-
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>
+
sufficient to show
-
 
+
the functionality of the CD </span><span lang="EN-US">(Fuchita
-
<div align=center>
+
et al. 2009b)</span><span lang="EN-US">. As
-
 
+
demonstrated in </span><span lang="EN-US">Figure 11</span><span
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0
+
lang="EN-US"> cytotoxicity of 5-FC is remarkable.</span></p>
-
  style='border-collapse:collapse;border:none'>
+
<div align="center">
-
<tr style='height:351.0pt'>
+
<table class="MsoTableGrid"
-
  <td width=610 valign=top style='width:457.65pt;padding:0cm 5.4pt 0cm 5.4pt;
+
  style="border: medium none ; border-collapse: collapse;"
-
  height:351.0pt'>
+
border="0" cellpadding="0" cellspacing="0">
-
  <p class=MsoNormal style='text-indent:0cm;page-break-after:avoid'><img
+
  <tbody>
-
  width=606 height=421 id="Diagramm 5"
+
    <tr style="height: 351pt;">
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image017.png"></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 lang=EN-US style='font-weight:
+
valign="top" width="610">
-
  normal'>Transduction of HT1080 with cytosine deaminase-packed viral
+
      <p class="MsoNormal"
-
  particles. 5-FC: 5-fluorocytosine (53 mM)</span></p>
+
style="text-indent: 0cm; page-break-after: avoid;"><img
-
  </td>
+
style="width: 606px; height: 421px;" alt=""
-
</tr>
+
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>
</table>
-
 
</div>
</div>
-
 
+
<p class="MsoNormal" style="text-indent: 0cm;"><span
-
<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+
lang="EN-US">&nbsp;</span></p>
-
 
+
<p class="MsoNormal"><span lang="EN-US">After
-
<p class=MsoNormal><span lang=EN-US>After three days of incubation in 5-fluorocytosine,
+
three days of incubation in 5-fluorocytosine,
-
the cells were washed, detached with Trypsin, centrifuged at 200 g for 5 min
+
the cells were washed, detached with Trypsin, centrifuged at 200 g for
-
followed by two washing steps with PBS and finally resuspended with 200 µl
+
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.</span></p>
DMEM. Living cells were then counted via Trypan blue staining.</span></p>
-
 
+
<p class="MsoNormal"><span lang="EN-US">After
-
<p class=MsoNormal><span lang=EN-US>After this successful qualitative demonstration
+
this successful qualitative demonstration
-
of an AAV2-mediated cytosine deaminase treatment, the bystander effect was quantified
+
of an AAV2-mediated cytosine deaminase treatment, the bystander effect
-
as well. The activated 5-FC molecules are able to diffuse through the plasma
+
was quantified
 +
as well. The activated 5-FC molecules are able to diffuse through the
 +
plasma
membrane and effect cells that are not transduced (</span><span
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
+
lang="EN-US">Figure 12</span><span lang="EN-US">).
-
untransduced HT1080 cells, which were mixed with the CD-transduced HT1080 cells</span><span
+
The bystander effect was tested with
-
lang=EN-US>.</span></p>
+
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"
-
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0
+
  style="border: medium none ; border-collapse: collapse;"
-
  style='border-collapse:collapse;border:none'>
+
border="0" cellpadding="0" cellspacing="0">
-
<tr>
+
  <tbody>
-
  <td width=614 valign=top style='width:460.6pt;padding:0cm 5.4pt 0cm 5.4pt'>
+
    <tr>
-
  <p class=MsoNormal align=left style='text-align:left;text-indent:0cm;
+
      <td style="padding: 0cm 5.4pt; width: 460.6pt;"
-
  page-break-after:avoid'><img width=607 height=478 id="Diagramm 1158"
+
valign="top" width="614">
-
  src="Arming%20-%20Killing%20the%20tumor_Uploaded%20on%20wiki_FINAL_1-Dateien/image018.png"></p>
+
      <p class="MsoNormal"
-
  <p class=MsoCaption align=left style='text-align:left'><a name="_Ref275979941"><span
+
style="text-align: left; text-indent: 0cm; page-break-after: avoid;"
-
  lang=EN-US>Figure </span></a><span lang=EN-US>12</span><span lang=EN-US>: </span><span
+
align="left"><img style="width: 607px; height: 478px;"
-
  lang=EN-US style='font-weight:normal'>Quantifying the bystander effect on
+
alt="" id="Diagramm 1158"
-
  HT1080 cells  5-FC: 5-fluorocytosine (53 mM)</span></p>
+
src="http://2010.igem.org/wiki/images/8/8c/Freiburg10_CD_Bystander.png"></p>
-
  </td>
+
      <p class="MsoCaption" style="text-align: left;"
-
</tr>
+
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>
</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
-
<p class=MsoNormal><span lang=EN-US> To prevent systemic toxic side effects of
+
prevent systemic toxic side effects of
-
conventional chemotherapy the iGEM team Freiburg_Bioware 2010 took a leap and
+
conventional chemotherapy the iGEM team Freiburg_Bioware 2010 took a
-
efficiently retargeted the viral vector for directed suicide gene delivery
+
leap and
-
towards tumor cells. Capsid engineering was successfully demonstrated by the
+
efficiently retargeted the viral vector for directed suicide gene
-
iGEM team Freiburg_Bioware 2010. Further details can be found under Results –
+
delivery
 +
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:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+
lang="EN-US">Ardiani, A., Sanchez-Bonilla, M. &amp;
-
font-family:"Calibri","sans-serif"'>Ardiani, A., Sanchez-Bonilla, M. &amp;
+
Black, M.E., 2010. Fusion enzymes containing HSV-1 thymidine kinase
-
Black, M.E., 2010. Fusion enzymes containing HSV-1 thymidine kinase mutants and
+
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
-
<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-
font-family:"Calibri","sans-serif"'>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
-
<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-
font-family:"Calibri","sans-serif"'>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
-
<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-
font-family:"Calibri","sans-serif"'>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
-
69(11), 4791-9. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2765227&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
+
5-fluorocytosine-mediated cell killing in vitro and in vivo. <i>Cancer
-
 
+
research</i>,
-
<p style='text-indent:36.0pt'><span style='font-size:10.0pt;font-family:"Calibri","sans-serif"'>Greco,
+
69(11), 4791-9. Available at:
-
O. &amp; Dachs, G.U., 2001. </span><span lang=EN-US style='font-size:10.0pt;
+
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2765227&amp;tool=pmcentrez&amp;rendertype=abstract.</span></p>
-
font-family:"Calibri","sans-serif"'>Gene directed enzyme/prodrug therapy of
+
<p style="text-indent: 36pt;"><span
-
cancer: historical appraisal and future prospectives. <i>Journal of cellular
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Greco,
 +
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
 +
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
-
<p style='text-indent:36.0pt'><span style='font-size:10.0pt;font-family:"Calibri","sans-serif"'>Huber,
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Huber,
-
B.E. et al., 1993. </span><span lang=EN-US style='font-size:10.0pt;font-family:
+
B.E. et al., 1993. </span><span
-
"Calibri","sans-serif"'>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
-
<p style='text-indent:36.0pt'><span style='font-size:10.0pt;font-family:"Calibri","sans-serif"'>Huber,
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Huber,
-
B.E. et al., 1994. </span><span lang=EN-US style='font-size:10.0pt;font-family:
+
B.E. et al., 1994. </span><span
-
"Calibri","sans-serif"'>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
 +
of the United States of America</i>, 91(17), 8302-6. Available
 +
at:
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>
-
 
+
<p style="text-indent: 36pt;"><span
-
<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-
font-family:"Calibri","sans-serif"'>Moolten, F.L., 1986. Tumor chemosensitivity
+
lang="EN-US">Moolten, F.L., 1986. Tumor chemosensitivity
-
conferred by inserted herpes thymidine kinase genes: paradigm for a prospective
+
conferred by inserted herpes thymidine kinase genes: paradigm for a
-
cancer control strategy. <i>Cancer research</i>, 46(10), 5276-81. Available at:
+
prospective
 +
cancer control strategy. <i>Cancer research</i>, 46(10),
 +
5276-81. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/3019523.</span></p>
http://www.ncbi.nlm.nih.gov/pubmed/3019523.</span></p>
-
 
+
<p style="text-indent: 36pt;"><span
-
<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-
font-family:"Calibri","sans-serif"'>Roche, <i>Apoptosis , Cell Death and Cell
+
lang="EN-US">Roche, <i>Apoptosis , Cell Death and
 +
Cell
Proliferation</i>,</span></p>
Proliferation</i>,</span></p>
-
 
+
<p style="text-indent: 36pt;"><span
-
<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-
font-family:"Calibri","sans-serif"'>Willmon, C.L., Krabbenhoft, E. &amp; Black,
+
lang="EN-US">Willmon, C.L., Krabbenhoft, E. &amp; Black,
-
M.E., 2006. A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances
+
M.E., 2006. A guanylate kinase/HSV-1 thymidine kinase fusion protein
-
prodrug-mediated cell killing. <i>Gene therapy</i>, 13(17), 1309-12. Available
+
enhances
 +
prodrug-mediated cell killing. <i>Gene therapy</i>,
 +
13(17), 1309-12. Available
at: http://www.ncbi.nlm.nih.gov/pubmed/16810197.</span></p>
at: http://www.ncbi.nlm.nih.gov/pubmed/16810197.</span></p>
-
 
+
<p style="text-indent: 36pt;"><span lang="EN-US">&nbsp;</span></p>
-
<p style='text-indent:36.0pt'><span lang=EN-US>&nbsp;</span></p>
+
-
 
+
</div>
</div>
 +
</body>
</html>
</html>
 +
{{:Team:Freiburg_Bioware/Footer}}
{{:Team:Freiburg_Bioware/Footer}}

Revision as of 21:27, 27 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. The 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

Assembly of the constructs carrying the suicide genes (termed vector plasmids) 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 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).

 

In order to modularize 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 pre- and suffix restriction sites. Figure 1 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 (Figure 2).

Figure 2: 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.

 

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 (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 DNA was be successfully ligated. Each assembly step for producing BioBricks was conducted following the iGEM BioBrick standard.

Figure 3: Cloning of the composite parts mGMK_TK30 to hGH-terminator_rightITR (insert). The digested fragments 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 in presence and absence of ganciclovir. Morphological changes were monitored via phase-contrast microscopy until 48 hours post infection. As it can be seen in Figure 4 non-transduced cells treated with ganciclovir and transduced cell without ganciclovir did not show significant tumor 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 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.

 

Suicide gene therapy is based on the conversion of non-toxic prodrugs to toxic substances (Greco & Dachs 2001), leading to cell death (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 over the suicide gene therapy approach. Non-toxic prodrugs are converted into toxic effector molecules leading to cell death of the tumor cells.

 

 

Since ganciclovir is not toxic for cells, non-transduced cells can survive in the presence of the prodrug (Figure 4A). 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.

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 binds specifically phosphatidylserine which is only accessible during apoptosis. Figure 6 demonstrates the relation between cell death and ganciclovir concentration.

 

Figure 6: 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.

 

 

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 led to significant cell death.

 

Effect of different ganciclovir concentrations on transduced HT1080 sarcoma cells. Transduction has been 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 higher the ganciclovir concentration, the more transduced cells were killed.

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 tetrazole, which is reduced to purple insoluble formazan in the presence of NADH and NADPH (Roche n.d.). The 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 treated with ganciclovir. 48 and 72 hours post infection cells were incubated with MTT and absorbance of formazan was quantified.

A

B

Figure 8: 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.

 

A

B

Figure 9: 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.

 

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 Untransduced 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 treating cancer. 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 mVenus 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 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.