Difference between revisions of "Part:BBa K404122"

Revision as of 00:17, 28 October 2010

[AAV2]-left-ITR_pCMV_betaglobin_mGMK_TK30_hGH_[AAV2]-right-ITR

This composite BioBrick serves as an example for the possibilities of the Virus Construction Kit created by the iGEM Team Freiburg Bioware 2010. The has been assembled from 7 single BioBricks: A promoter, a transcription-enhancing sequence, a Gene of Interest that is itself a fusion protein, a polyadenylation sequence required for proper translation and inverted terminal repeats that are required for replication at both ends of the DNA molecule. Cotransfected into eukaryotic helper cell lines together with a plasmid encoding AAV Rep and Cap proteins and a pHelper plasmid encoding essential adenoviral genes, this BioBrick is being packaged into AAV capsids in form of a single-stranded DNA molecule and hence substitutes the AAV genome. AAV particles containing this Biobrick as a vector plasmid are replication-deficient and are therefore considered as biosafety level 1.

Freiburg10 Vectorplasmid composite 4.png

Functional Composite Part for Successfull Tumor Cell Ablation

Introduction

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

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

Successful Assembly of Vector Plasmids Carrying Suicide Genes via Cloning

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

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

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

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

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

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

Monitoring Efficient Tumor Killing by Phase-Contrast Microscopy

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

A Beschreibung: https://static.igem.org/mediawiki/2010/3/3e/HT_negativ_control_ganciclovir_only.jpg

B Beschreibung: https://static.igem.org/mediawiki/2010/f/f0/HT_TKGMK_clone_1_ohne_Ganciclovir_300%C2%B5l.jpg

C

Beschreibung: https://static.igem.org/mediawiki/2010/c/c3/HT_TKGMK_clone_1_with_ganciclovir_300%C2%B5l.jpg

D Beschreibung: https://static.igem.org/mediawiki/2010/7/73/HT_TKGMK_clone_1_with_ganciclovir_600%C2%B5l_well_2.jpg

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

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

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

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

Quantitative Analysis of Cell Death by Flow Cytometry

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

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

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

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

Titrating Ganciclovir Concentrations for Efficient Cell Killing by Cytotoxicity Assays

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

A

B

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

A

B

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

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

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

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 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 iGEM wiki Freiburg_Bioware 2010 - 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.

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.

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.

ITRs

The inverted terminal repeat structures can be subdivided into several palindromic motives: A and A’ form a stem loop which encases B and B’ as well as C and C’. Those motives form both arms of the T-shaped structure. The functional motives on the ITR are two regions that bind Rep 68/78, called Rep-binding elements (RBE on the stem and RBE’ on the B arm) and the terminal resolution site (trs) in which the rep proteins introduce single-stranded nicks. The 3’ OH end of the A motive acts as a primer for DNA replication (Im & Muzyczka, 1990) (Lusby, Fife, & Berns, 1980).

CMV promoter

Beta-globin-intron

The beta-globin intron BioBrick consists of a partial chimeric CMV promoter followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 25 bases of human beta globin gene exon 3. The beta globin intron BioBrick is assumed to enhance eukaryotic gene expression (Nott, Meislin, & Moore, 2003). As shown in Figure 9 and Figure 10 the vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results and taking into account that a constant volume of viral particles has been used for transduction, the difference between the construct containing and lacking the beta-globin intron is minimal. Since packaging efficiency of the AAV-2 decreases with increasing sizes of the insert (Dong, Fan, & Frizzell, 1996), the iGEM team Freiburg_Bioware suggests using the beta-globin intron in dependence on the size of your transgene.

mGMK_TK30 fusion protein

The thymidine kinase mutant TK30 contains six modified amino acids (Black, Newcomb, Wilson, & Loeb, 1996) created in a first screening showing enhanced affinity for gancivlocir and acyclovir, but reduced specificity for its natural substrate thymidine.

As efficient tumor killing and therefore ganciclovir activation is essential for successful tumor ablation, further improvements were conducted. Overexpression of transgenic thymidine kinase leads to accumulation of non-toxic intermediates, which cannot be phosphorylated sufficiently by endogenous guanylate kinase, the second enzyme in the salvage pathway of nucleotides.
Overcoming this bottleneck was accomplished by fusing the mouse guanylate kinase (mGMK) to the N-terminus of TK30 mutant creating a fusion protein (mGMK_TK30) with enhanced GCV/ACV sensitivity in vitro and in vivo (Ardiani, Sanchez-Bonilla, & Black, 2010) and improved bystander activity. The effect of non-transfected tumor cell killing upon transfer of toxic metabolites through gap junctional intercellular communication (GJIC) or immune-mediated tumor ablation is essential in suicide gene therapy (Pope, 1997). GCV-triphosphate is mainly transported through the central pore formed between connexin proteins from neighboring cells (Gentry, Im, Boucher, Ruch, & Shewach, 2005), but immune-induced bystander effect seems to be likely as well (Grignet-Debrus, Cool, Baudson, Velu, & Calberg-Bacq, 2000).

hGH

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1319
Illegal NgoMIV site found at 1977
Illegal NgoMIV site found at 2882
Illegal AgeI site found at 3011
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 3413
Illegal SapI.rc site found at 1369

@@ Line 5: / Line 5: @@
 Cotransfected into eukaryotic helper cell lines together with a plasmid encoding AAV Rep and Cap proteins and a pHelper plasmid encoding essential adenoviral genes, this BioBrick is being packaged into AAV capsids in form of a single-stranded DNA molecule and hence substitutes the AAV genome.
 AAV particles containing this Biobrick as a vector plasmid are replication-deficient and are therefore considered as biosafety level 1.
+[[Image:Freiburg10_Vectorplasmid composite 4.png|thumb|center|480px]]<br>
 <html>
-<p class="MsoTocHeading">Contents</p>
+<h2>Contents</h2>
-<p class="MsoToc2"><a href="#_Toc275981841"><span lang="EN-US">Arming: Suicide
+<p class="MsoToc2"><a href="#_Toc275981841"><span lang="EN-US">Functional Composite Part for Successfull Tumor Cell Ablation</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">1</span></a></p>
-Genes as GOIs</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">1</span></a></p>
 <p class="MsoToc3"><a href="#_Toc275981842"><span lang="EN-US">Introduction</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">1</span></a></p>
@@ Line 26: / Line 27: @@
 Ganciclovir Concentrations for Efficient Cell Killing by Cytotoxicity Assays</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">8</span></a></p>
-<p class="MsoToc3"><a href="#_Toc275981847"><span lang="EN-US">Killing Untransduced
-Tumor Cells via Bystander Effect</span><span style="color: windowtext; display: none; text-decoration: none;"> </span><span style="color: windowtext; display: none; text-decoration: none;">13</span></a></p>
 <p class="MsoToc3"><a href="#_Toc275981848"><span lang="EN-US">Conclusions</span><span style="color: windowtext; display: none; text-decoration: none;">. </span><span style="color: windowtext; display: none; text-decoration: none;">16</span></a></p>
@@ Line 35: / Line 34: @@
 <p class="MsoNormal">&nbsp;</p>
-<h2><a name="_Toc275981841"><span lang="EN-US">Arming: Suicide Genes as GOIs</span></a></h2>
+<h2><a name="_Toc275981841"><span lang="EN-US">Functional Composite Part for Successfull Tumor Cell Ablation</span></a></h2>
 <h3><a name="_Toc275981842"><span lang="EN-US">Introduction</span></a></h3>
@@ Line 376: / Line 375: @@
 was plotted against increasing ganciclovir concentrations. </span></p>
-<h3><a name="_Toc275981847"><span lang="EN-US">Killing Non-transduced Tumor Cells
-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
-convertase negative cells surrounded by suicide enzyme positive cells did not
-survive prodrug treatment. Besides efficient killing of targeted tumor cells,
-neighboring, non-transduced cells are killed as well, providing an important
-effect in cancer treatment. Since 5-Fluorouracil is soluble and can diffuse
-into adjacent cells </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>
-<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<div align="center">
-<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0">
- <tbody><tr>
-  <td style="padding: 0cm 5.4pt; width: 460.6pt;" valign="top" width="614">
-  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 614px; height: 262px;" alt="" id="Grafik 82" src="https://static.igem.org/mediawiki/2010/c/c0/Freiburg10_Bystander_effect.png"></p>
-  <p class="MsoCaption"><span lang="EN-US">Figure </span><span lang="EN-US">10</span><span lang="EN-US">: Schematic overview of the Bystander
-  effect.</span></p>
-  </td>
- </tr>
-</tbody></table>
-</div>
-<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
-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:</span></p>
-<p class="MsoListParagraphCxSpFirst" style="margin-left: 53.85pt; text-indent: -18pt;"><span style="font-family: Symbol;">·<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-</span></span>pHelper</p>
-<p class="MsoListParagraphCxSpMiddle" style="margin-left: 53.85pt; text-indent: -18pt;"><span style="font-family: Symbol;" lang="EN-US">·<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-</span></span><span lang="EN-US">Rep/Cap:
-pSB1C3_001_[AAV2]-Rep-VP123(ViralBrick-587KO-empty)_p5-TATAless</span></p>
-<p class="MsoListParagraphCxSpLast" style="margin-left: 53.85pt; text-indent: -18pt;"><span style="font-family: Symbol;" lang="EN-US">·<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-</span></span><span lang="EN-US">Gene of interest:
-pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_CD_hGH_[AAV2]-right-ITR</span></p>
-<p class="MsoNormal"><span lang="EN-US">90 % confluent HT1080 cells were transduced
-in T75 flasks with 9 ml of viral stock. In parallel, another T75 flask was
-transduced with a viral stock packaged with the mVenus coding sequence to
-assess transgene expression.</span></p>
-<p class="MsoNormal"><span lang="EN-US">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.</span></p>
-<p class="MsoNormal"><span lang="EN-US">The incubation with the prodrug
--fluorocytosine (5-FC) was performed at a final concentration of 53 mM.
-According to Fuchita <i>et al.,</i> this amount should be sufficient to show
-the functionality of the CD </span><span lang="EN-US">(Fuchita et al. 2009b)</span><span lang="EN-US">. As demonstrated in </span><span lang="EN-US">Figure 11</span><span lang="EN-US">, cytotoxicity of 5-FC is
-remarkable.</span></p>
-<div align="center">
-<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0">
- <tbody><tr style="height: 351pt;">
-  <td style="padding: 0cm 5.4pt; width: 457.65pt; height: 351pt;" valign="top" width="610">
-  <p class="MsoNormal" style="text-indent: 0cm; page-break-after: avoid;"><img style="width: 610px; height: 424px;" alt="" src="https://static.igem.org/mediawiki/2010/4/4c/Freiburg10_CD_ToxicEffect.png" id="Diagramm 5"></p>
-  <p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure </span><span lang="EN-US">11</span><span lang="EN-US">: </span><span style="font-weight: normal;" lang="EN-US">Transduction of HT1080 with cytosine deaminase-packed viral
-  particles. 5-FC: 5-fluorocytosine (53 mM)</span></p>
-  </td>
- </tr>
-</tbody></table>
-</div>
-<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<p class="MsoNormal"><span lang="EN-US">After three days of incubation in 5-fluorocytosine,
-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.</span></p>
-<p class="MsoNormal"><span lang="EN-US">After this successful qualitative
-demonstration of an AAV2-mediated cytosine deaminase treatment, the bystander
-effect was quantified as well. The activated 5-FC molecules are able to diffuse
-through the plasma membrane and thus effect cells that are not transduced (</span><span lang="EN-US">Figure 12</span><span lang="EN-US">). The bystander effect was tested with
-untransduced HT1080 cells, which were mixed with the CD-transduced HT1080 cells</span><span lang="EN-US">.</span></p>
-<div align="center">
-<table class="MsoTableGrid" style="border: medium none ; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0">
- <tbody><tr>
-  <td style="padding: 0cm 5.4pt; width: 460.6pt;" valign="top" width="614">
-  <p class="MsoNormal" style="text-align: left; text-indent: 0cm; page-break-after: avoid;" align="left"><img style="width: 614px; height: 483px;" alt="" id="Diagramm 1158" src="https://static.igem.org/mediawiki/2010/8/8c/Freiburg10_CD_Bystander.png"></p>
-  <p class="MsoCaption" style="text-align: left;" align="left"><a name="_Ref275979941"><span lang="EN-US">Figure </span></a><span lang="EN-US">12</span><span lang="EN-US">: </span><span style="font-weight: normal;" lang="EN-US">Quantifying the bystander effect on
-  HT1080 cells&nbsp; 5-FC: 5-fluorocytosine (53 mM)</span></p>
-  </td>
- </tr>
-</tbody></table>
-</div>
-<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<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 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.</span></p>
 <h3><a name="_Toc275981848"><span lang="EN-US">Conclusions</span></a></h3>
@@ Line 498: / Line 385: @@
 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
+caused by the introduction of modified fusion genes
 consisting of guanylate and thymidine kinases.</span></p>
@@ Line 505: / Line 392: @@
 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 –
+iGEM team Freiburg_Bioware 2010. Further details can be found under  <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results#targeting">iGEM wiki Freiburg_Bioware 2010 - Results - Targeting</a>
-Targeting.</span></p>
+</span></p>
 <h3><a name="_Toc275981849"><span lang="EN-US">References</span></a></h3>
@@ Line 546: / Line 433: @@
 http://www.ncbi.nlm.nih.gov/pubmed/8402637.</span></p>
-<p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Huber,
-B.E. et al., 1994. </span><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Metabolism of 5-fluorocytosine to 5-fluorouracil in
-human colorectal tumor cells transduced with the cytosine deaminase 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>
 <p style="text-indent: 36pt;"><span style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;" lang="EN-US">Moolten, F.L., 1986. Tumor chemosensitivity
@@ Line 570: / Line 450: @@
 </html>
-[[Image:Freiburg10_Vectorplasmid composite 4.png|thumb|center|480px]]<br>
 <h2>ITRs</h2><br>
 <html>