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

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Contents

Introduction to Modularization of Vectorplasmid. 1

Recombinant and Modular Vectorplasmid Carrying GOI 2

Cloning and Combination Strategies for the Vectorplasmid. 3

Testing functionality of Assembled Vectorplasmid. 7

Fluorescence Microscopy of Target Cells Demonstrates GOI Expression. 7

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

Influence of hGH terminator BioBrick on GOI Expression. 8

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

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

Conclusion. 16

References. 17

Introduction to Modularization of Vectorplasmid

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

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

Recombinant and Modular Vectorplasmid Carrying GOI

The iGEM team Freiburg_Bioware 2010 provides a modular Virus Construction Kit for therapeutical applications, quantification assays and purification approaches depending on capsid modifications and the gene of interest flanked by the inverted terminal repeats (ITRs. In order to produce BioBrick-compatible standardized biological parts, we reengineered the plasmids and added new components for gene therapy approaches and analysis of biological activity of assembled BioBrick parts. Each element required for intact and functional plasmids comprising the ITRs, a promoter, a putative enhancer element and the hGH terminator was PCR amplified and fused together de novo. As shown in Figure 1, the vectorplasmid was assembled with the produced BioBricks consisting of the left and right ITR (BBa_K404100 and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin intron (BBa_K404107), the gene of interests (fluorescent proteins mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).

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

Cloning and Combination Strategies for the Vectorplasmid

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Testing functionality of Assembled Vectorplasmid

Fluorescence Microscopy of Target Cells Demonstrates GOI Expression

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

A

Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)

B

Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG

C

Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg

D

Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg

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

Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression

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

Influence of hGH terminator BioBrick on GOI Expression

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

Vectorplasmid lacking hGH termination signal

Vectorplasmid containing hGH terminator signal

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

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

Influence of Beta-globin intron Biobrick on GOI Expression

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

Vectorplasmid lacking beta-globin intron

Vectorplasmid containing beta-globin intron

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

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

Functionality of the Full Assembled Vectorplasmid Demonstrated by GOI Expression

After assembly of plasmids containing all required elements (see Figure 1), functionality was tested in cell culture. AAV-293 cells stably expressing E1A and E1B proteins were transfected with three plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72-hours post-transfection and the tumor cell line HT1080 was transduced with the recombinant viral vectors encapsidating the gene of interest mVenus (BBa_I757008).

The iGEM team Freiburg_Bioware 2010 compared the standard-plasmid containing a subcloned mVenus (pAAV_mVenus, derived from the Stratagene system) with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by flow cytometry analysis are shown in Figure 11 and Figure 12. Comparing mVenus expression of the standard plasmid and the modified, assembled plasmid reveals that biological functionality of the reassembled plasmid was confirmed.

pSB1C3_mVenus (BBa_K404119)

pAAV_mVenus (Stratagene)

Figure 12: Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to standard plasmid provided by Stratagene. A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells plotted against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5).C: Gating transduced cells (R2 ≙R14) (used plasmids for transfection: pGOI: pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119), pHelper, pRC. D: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression. E: Overlay of non-transduced (red) and transduced (green). F: Gating non transduced cells (control). G: Non-transduced cells plotted against mVenus (R5). H: Gating transduced cells (R14 ≙R2) (used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). I: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells plotted against mVenus expression.

Figure 13: Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to standard plasmid provided by Stratagene. Fluorescence of the standard plasmid pAAV_mVenus (Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct was measured. As demonstrated mVenus expression is enhanced in the assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus construct.

Conclusion

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

References

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

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

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

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

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-style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
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+style='color:windowtext;display:none;text-decoration:none'>1</span></a></span></p>
-lang="EN-US">Recombinant and Modular Vectorplasmid Carrying GOI</span><span
-style="color: windowtext; display: none; text-decoration: none;"> </span><span
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-style="color: windowtext; display: none; text-decoration: none;">2</span></a></span></p>
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-<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800682"><span
+style='color:windowtext;display:none;text-decoration:none'> </span><span
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-<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800683"><span
+lang=EN-US>Cloning and Combination Strategies for the Vectorplasmid</span><span
-lang="EN-US">Testing functionality of Assembled Vectorplasmid</span><span
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
-style="color: windowtext; display: none; text-decoration: none;">. </span><span
+style='color:windowtext;display:none;text-decoration:none'>3</span></a></span></p>
-style="color: windowtext; display: none; text-decoration: none;">7</span></a></span></p>
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-lang="EN-US">Fluorescence Microscopy of Target Cells Demonstrates GOI
+lang=EN-US>Testing functionality of Assembled Vectorplasmid</span><span
-Expression</span><span
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
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+style='color:windowtext;display:none;text-decoration:none'>7</span></a></span></p>
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-lang="EN-US">Analysis of Target Cells by Flow Cytometry demonstrates
+lang=EN-US>Fluorescence Microscopy of Target Cells Demonstrates GOI Expression</span><span
-GOI Expression</span><span
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
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-lang="EN-US">Influence of hGH terminator BioBrick on GOI Expression</span><span
+lang=EN-US>Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression</span><span
-style="color: windowtext; display: none; text-decoration: none;">. </span><span
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
-style="color: windowtext; display: none; text-decoration: none;">8</span></a></span></p>
+style='color:windowtext;display:none;text-decoration:none'>8</span></a></span></p>
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-lang="EN-US">Influence of <i>Beta-globin</i> intron Biobrick on GOI
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-Expression</span><span
+lang=EN-US>Influence of hGH terminator BioBrick on GOI Expression</span><span
-style="color: windowtext; display: none; text-decoration: none;">. </span><span
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
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-<p class="MsoToc5"><span class="MsoHyperlink"><a href="#_Toc275800688"><span
-lang="EN-US">Functionality of the Full Assembled Vectorplasmid
+<p class=MsoToc5><span class=MsoHyperlink><a href="#_Toc275800687"><span
-Demonstrated by
+lang=EN-US>Influence of <i>Beta-globin</i> intron Biobrick on GOI Expression</span><span
-GOI Expression</span><span
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
-style="color: windowtext; display: none; text-decoration: none;">. </span><span
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-style="color: windowtext; display: none; text-decoration: none;">14</span></a></span></p>
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-lang="EN-US">Conclusion</span><span
+lang=EN-US>Functionality of the Full Assembled Vectorplasmid Demonstrated by
-style="color: windowtext; display: none; text-decoration: none;">. </span><span
+GOI Expression</span><span style='color:windowtext;display:none;text-decoration:
-style="color: windowtext; display: none; text-decoration: none;">16</span></a></span></p>
+none'>. </span><span
-<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800690"><span
+style='color:windowtext;display:none;text-decoration:none'>14</span></a></span></p>
-lang="EN-US">References</span><span
-style="color: windowtext; display: none; text-decoration: none;">. </span><span
+<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275800689"><span
-style="color: windowtext; display: none; text-decoration: none;">17</span></a></span></p>
+lang=EN-US>Conclusion</span><span style='color:windowtext;display:none;
-<p class="MsoNormal"><a name="_Toc275797953"><b>&nbsp;</b></a></p>
+text-decoration:none'>. </span><span
-<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800680">Introduction
+style='color:windowtext;display:none;text-decoration:none'>16</span></a></span></p>
-to Modularization of Vectorplasmid</a></h2>
-<p class="MsoNormal"><span lang="EN-US">Producing recombinant virus
+<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275800690"><span
-particles for
+lang=EN-US>References</span><span style='color:windowtext;display:none;
-therapeutical means is, besides specifically target cells, purification
+text-decoration:none'>. </span><span
-and
+style='color:windowtext;display:none;text-decoration:none'>17</span></a></span></p>
-quantification assays of AAV-2, one intention of the Virus Construction
-Kit
+<p class=MsoNormal><a name="_Toc275797953"><b>&nbsp;</b></a></p>
-provided by the iGEM team Freiburg_Bioware 2010. For obtaining a
-modular
+<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800680">Introduction to Modularization of Vectorplasmid</span></a></h2>
-toolkit, the complex components of AAV-2 were extracted and redesigned
-to match
+<p class=MsoNormal><span lang=EN-US>Producing recombinant virus particles for
+therapeutical means is, besides specifically target cells, purification and
+quantification assays of AAV-2, one intention of the Virus Construction Kit
+provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular
+toolkit, the complex components of AAV-2 were extracted and redesigned to match
 the iGEM standard. Functional activity was tested in cell culture.</span></p>
-<p class="MsoNormal"><span lang="EN-US">Differing from the wildtype
-AAV-2 genome,
+<p class=MsoNormal><span lang=EN-US>Differing from the wildtype AAV-2 genome,
-the Helper Free System provided by Stratagene comprises three plasmids
+the Helper Free System provided by Stratagene comprises three plasmids and a
-and a
+specialized production cell line. AAV-293 cells derived from the HEK cell line
-specialized production cell line. AAV-293 cells derived from the HEK
+express the stably integrated E1A and E1B helper proteins for efficient virus
-cell line
+production. The plasmid containing the inverted terminal repeats (ITRs) is
-express the stably integrated E1A and E1B helper proteins for efficient
+encapsidated into the preformed capsids after production of single-stranded DNA
-virus
-production. The plasmid containing the inverted terminal repeats (ITRs)
-is
-encapsidated into the preformed capsids after production of
-single-stranded DNA
 therefore also known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i>
-intron and the hGH terminator signal are flanked by the ITRs and serve
+intron and the hGH terminator signal are flanked by the ITRs and serve in the
-in the
+host cell for regulation of transgene expression. In addition to that, the
-host cell for regulation of transgene expression. In addition to that,
+plasmid coding for the Rep and Cap proteins (pRC) can be provided <i>in trans</i>
-the
+leading to a layer of specificity due to the fact that the two genes are not
-plasmid coding for the Rep and Cap proteins (pRC) can be provided <i>in
+packaged into the capsid since lacking of the ITRs impairs encapsidation. Another
-trans</i>
+advantage of the Helper Free System can be attributed to cotransfection of
-leading to a layer of specificity due to the fact that the two genes
-are not
-packaged into the capsid since lacking of the ITRs impairs
-encapsidation. Another
-advantage of the Helper Free System can be attributed to cotransfection
-of
 another helper plasmid (pHelper), which provides the necessary proteins
-normally obtained by superinfection with helper viruses such as
+normally obtained by superinfection with helper viruses such as adenovirus or
-adenovirus or
+herpes simplex virus. These helper genes are required for full viral assembly
-herpes simplex virus. These helper genes are required for full viral
-assembly
 by regulating gene expression of Rep and Cap proteins.</span></p>
-<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800681"></a><a
+<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800681"></a><a
 name="_Toc275797954">Recombinant and Modular Vectorplasmid Carrying
 GOI</a></h3>
-<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware
-provides
+<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware 2010 provides
-a modular Virus Construction Kit for therapeutical applications,
+a modular Virus Construction Kit for therapeutical applications, quantification
-quantification
+assays and purification approaches depending on capsid modifications and the
-assays and purification approaches depending on capsid modifications
+gene of interest flanked by the inverted terminal repeats (ITRs. In order to
-and the
+produce BioBrick-compatible standardized biological parts, we reengineered the
-gene of interest flanked by the inverted terminal repeats (ITRs. In
+plasmids and added new components for gene therapy approaches and analysis of
-order to
+biological activity of assembled BioBrick parts. Each element required for
-produce BioBrick-compatible standardized biological parts, we
+intact and functional plasmids comprising the ITRs, a promoter, a putative
-reengineered the
+enhancer element and the hGH terminator was PCR amplified and fused together <i>de
-plasmids and added new components for gene therapy approaches and
+novo</i>.</span><span lang=EN-US> As shown in </span><span
-analysis of
+lang=EN-US>Figure 1</span><span lang=EN-US>, the vectorplasmid was assembled
-biological activity of assembled BioBrick parts. Each element required
+with the produced BioBricks consisting of the left and right ITR (BBa_K404100
-for
+and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the
-intact and functional plasmids comprising the ITRs, a promoter, a
+beta-globin intron (BBa_K404107), the gene of interests (fluorescent proteins
-putative
+mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112,
-enhancer element and the hGH terminator was PCR amplified and fused
+mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).</span></p>
-together <i>de
-novo</i>.</span><span lang="EN-US"> As shown in </span><span
+<div align=center>
-lang="EN-US">Figure 1</span><span lang="EN-US">, the vectorplasmid was
-assembled
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-with the produced BioBricks consisting of the left and right ITR
+ style='border-collapse:collapse;border:none'>
-(BBa_K404100
+ <tr style='height:522.25pt'>
-and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT:
+  <td width=589 valign=top style='width:441.75pt;border:solid windowtext 1.0pt;
-BBa_K404106)) , the
+  padding:0cm 5.4pt 0cm 5.4pt;height:522.25pt'>
-beta-globin intron (BBa_K404107), the gene of interests (fluorescent
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-proteins
+cm;line-height:normal;page-break-after:avoid'><img width=580 height=692
-mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30:
+  id="Grafik 5" src="Freiburg10_Modularization_GOI-Dateien/image001.jpg"></p>
-BBa_K404112,
+  <p class=MsoCaption style='text-indent:0cm'><span lang=EN-US>Figure </span><span lang=EN-US>1</span><span lang=EN-US>: </span><span lang=EN-US style='font-weight:
-mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator
+  normal'>Overview of the theoretical sequence of each BioBrick provided within
-(BBa_K404108).</span></p>
+  the Virus Construction Kit for an intact and fully functional rAAV genome.
-<div align="center">
+  The plasmid in the lowest panel was used for tumor killing in combination with
-<table class="MsoTableGrid"
+  plasmids coding for modified capsid proteins. More detailed infomartion about
-style="border: medium none ; border-collapse: collapse;" border="1"
+  these constructs can be found under ‘Arming: Killing the tumor’ and
-cellpadding="0" cellspacing="0">
+  ‘N-terminal fusion for Targeting’.</span></p>
-<tbody>
+  </td>
-<tr style="height: 522.25pt;">
+ </tr>
-<td
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 441.75pt; height: 522.25pt;"
-valign="top" width="589">
-<p class="MsoNormal"
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-id="Grafik 5" src="Freiburg10_Modularization_GOI-Dateien/image001.jpg"
-height="692" width="580"></p>
-<p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure
-</span><span lang="EN-US">1</span><span lang="EN-US">: </span><span
-style="font-weight: normal;" lang="EN-US">Overview of the theoretical
-sequence of each BioBrick provided within the Virus Construction Kit
-for an intact and fully functional rAAV genome. The plasmid in the
-lowest panel was used for tumor killing in combination with plasmids
-coding for modified capsid proteins. More detailed infomartion about
-these constructs can be found under ‘Arming: Killing the tumor’ and
-‘N-terminal fusion for Targeting’.</span></p>
-</td>
-</tr>
-</tbody>
 </table>
 </div>
-<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<h3><a name="_Toc275800682"></a><a name="_Toc275797955"><span
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
-lang="EN-US">Cloning
-and Combination Strategies for the Vectorplasmid</span></a><span
+<h3><a name="_Toc275800682"></a><a name="_Toc275797955"><span lang=EN-US>Cloning
-lang="EN-US"> </span></h3>
+and Combination Strategies for the Vectorplasmid</span></a><span lang=EN-US> </span></h3>
-<p class="MsoNormal"><span lang="EN-US">Organization of the recombinant
-viral DNA was
+<p class=MsoNormal><span lang=EN-US>Organization of the recombinant viral DNA was
-modified ensuring several layers of specificity to our systems
+modified ensuring several layers of specificity to our systems including a
-including a
+tumor-specific promoter and suicide genes encoding prodrug convertases. In
-tumor-specific promoter and suicide genes encoding prodrug convertases.
+order to modularize the rAAV sequence, each plasmid element (</span><span lang=EN-US>Figure 1</span><span lang=EN-US>) was PCR-amplified and cloned into
-In
+the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team Freiburg_Bioware
-order to modularize the rAAV sequence, each plasmid element (</span><span
+performed three site-directed mutagenesis in the gene of interest TK30 (BBa_K404109)
-lang="EN-US">Figure 1</span><span lang="EN-US">) was PCR-amplified and
+and cytosine deaminase (</span><span lang=EN-US style='font-size:9.0pt;
-cloned into
+line-height:200%'>BBa_K404112</span><span lang=EN-US>) for deletion of PstI and
-the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team
+NgoMIV iGEM site (for further information see the results page of ‘Arming –
-Freiburg_Bioware
+Killing the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich
-performed three site-directed mutagenesis in the gene of interest
+regions forming T-shaped hairpins during replication, PCR amplification was not
-TK30 (BBa_K404109)
+possible. Hence a cloning strategy was developed by the iGEM team Freiburg in
-and cytosine deaminase (</span><span
+order to provide BioBrick-compatible ITRs (see ‘Method Development of Cloning
-style="font-size: 9pt; line-height: 200%;" lang="EN-US">BBa_K404112</span><span
-lang="EN-US">) for deletion of PstI and
-NgoMIV iGEM site (for further information see the results page of
-‘Arming –
-Killing the tumor’). Since the inverted terminal repeats (ITRs) are
-GC-rich
-regions forming T-shaped hairpins during replication, PCR amplification
-was not
-possible. Hence a cloning strategy was developed by the iGEM team
-Freiburg in
-order to provide BioBrick-compatible ITRs (see ‘Method Development of
-Cloning
 Strategy for ITRs’).</span></p>
-<p class="MsoNormal"><span lang="EN-US">In </span><span lang="EN-US">Figure
-</span><span lang="EN-US"> the schematic overview of the
+<p class=MsoNormal><span lang=EN-US>In </span><span
-modularization process can be seen which has been followed to conduct
+lang=EN-US>Figure 2</span><span lang=EN-US> the schematic overview of the
-the
+modularization process can be seen which has been followed to conduct the
 assembly steps required for functional vectorplasmids.</span></p>
-<div align="center">
-<table class="MsoTableGrid"
+<div align=center>
-style="border: medium none ; border-collapse: collapse;" border="1"
-cellpadding="0" cellspacing="0">
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-<tbody>
+ style='border-collapse:collapse;border:none'>
-<tr style="height: 26.15pt;">
+ <tr style='height:26.15pt'>
-<td
+  <td width=454 valign=top style='width:340.15pt;border:solid windowtext 1.0pt;
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"
+  padding:0cm 5.4pt 0cm 5.4pt;height:26.15pt'>
-valign="top" width="454">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<p class="MsoNormal"
+cm;line-height:normal;page-break-after:avoid'><img width=439 height=272
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+  id="Grafik 2" src="Freiburg10_Modularization_GOI-Dateien/image002.gif"
-id="Grafik 2" src="Freiburg10_Modularization_GOI-Dateien/image002.gif"
+  alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"></p>
-alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275783119"><span
-height="272" width="439"></p>
+  lang=EN-US>Figure </span></a><span lang=EN-US>3</span><span lang=EN-US>: </span><span
-<p class="MsoCaption" style="text-indent: 0cm;"><a
+  lang=EN-US style='font-weight:normal'>Assembly procedure for fusion of
-name="_Ref275783119"><span lang="EN-US">Figure </span></a><span
+  BioBricks and composite parts to a fully assembled and functional plasmid
-lang="EN-US">3</span><span lang="EN-US">: </span><span
+  coding for your gene of interest. This plasmid can be cotransfected with two
-style="font-weight: normal;" lang="EN-US">Assembly procedure for
+  helper plasmids providing protein for assembly and encapsidating of the rAAV
-fusion of BioBricks and composite parts to a fully assembled and
+  genome (your gene of interest) into the capsids.</span></p>
-functional plasmid coding for your gene of interest. This plasmid can
+  </td>
-be cotransfected with two helper plasmids providing protein for
+ </tr>
-assembly and encapsidating of the rAAV genome (your gene of interest)
-into the capsids.</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 iGEM team Freiburg_Bioware
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-provides two
-examples demonstrating the assembly procedure for constructing
+<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware provides two
-vectorplasmids.
+examples demonstrating the assembly procedure for constructing vectorplasmids.
 The first representative example is the fusion of the BioBrick part <i>beta-globin</i>
-to the composite parts containing the 5´ elements of the plasmids,
+to the composite parts containing the 5´ elements of the plasmids, which are
-which are
 left ITR and CMV or phTERT promoter, respectively.</span></p>
-<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">As
-shown in </span><span lang="EN-US">Figure 3</span><span lang="EN-US">
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>As shown in </span><span lang=EN-US>Figure 3</span><span lang=EN-US> the theoretical cloning performed
-the theoretical cloning performed
+for assembling the BioBricks <i>beta-globin </i>intron and leftITR_CMV together
-for assembling the BioBricks <i>beta-globin </i>intron and
-leftITR_CMV together
 can be observed. </span></p>
-<div align="center">
-<table class="MsoTableGrid"
+<div align=center>
-style="border: medium none ; border-collapse: collapse;" border="1"
-cellpadding="0" cellspacing="0">
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-<tbody>
+ style='border-collapse:collapse;border:none'>
-<tr style="height: 23.95pt;">
+ <tr style='height:23.95pt'>
-<td
+  <td width=467 valign=top style='width:349.9pt;border:solid windowtext 1.0pt;
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"
+  padding:0cm 5.4pt 0cm 5.4pt;height:23.95pt'>
-valign="top" width="467">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<p class="MsoNormal"
+cm;line-height:normal;page-break-after:avoid'><img width=452 height=530
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+  id="Grafik 74" src="Freiburg10_Modularization_GOI-Dateien/image003.gif"></p>
-id="Grafik 74" src="Freiburg10_Modularization_GOI-Dateien/image003.gif"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275783160"><span
-height="530" width="452"></p>
+  lang=EN-US>Figure </span></a><span lang=EN-US>4</span><span lang=EN-US>:</span><span
-<p class="MsoCaption" style="text-indent: 0cm;"><a
+  lang=EN-US> </span><span lang=EN-US style='font-weight:normal'>Theoretical
-name="_Ref275783160"><span lang="EN-US">Figure </span></a><span
+  cloning of the composite part leftITR_CMV to the <i>beta-globin</i> intron
-lang="EN-US">4</span><span lang="EN-US">:</span><span lang="EN-US"> </span><span
+  BioBrick leading to the plasmid leftITR_CMV_<i>beta-globin</i> intron.</span></p>
-style="font-weight: normal;" lang="EN-US">Theoretical cloning of the
+  </td>
-composite part leftITR_CMV to the <i>beta-globin</i> intron BioBrick
+ </tr>
-leading to the plasmid leftITR_CMV_<i>beta-globin</i> intron.</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">The plasmids were digested with
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
-both XbaI
-and PstI (beta-globin intron: </span><span
+<p class=MsoNormal><span lang=EN-US>The plasmids were digested with both XbaI
-style="font-size: 9pt; line-height: 200%; color: black;" lang="EN-US">BBa_K404107</span><span
+and PstI (beta-globin intron: </span><span lang=EN-US style='font-size:9.0pt;
-lang="EN-US">) or SpeI and
+line-height:200%;color:black'>BBa_K404107</span><span lang=EN-US>) or SpeI and
-PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the
+PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the preparative
-preparative
+gel in </span><span lang=EN-US>Figure 4</span><span lang=EN-US>, the expected
-gel in </span><span lang="EN-US">Figure 4</span><span lang="EN-US">,
-the expected
 bands could be detected under UV light and the extracted DNA could be
-successfully ligated. Each assembly step for producing BioBrick
+successfully ligated. Each assembly step for producing BioBrick intermediates
-intermediates
 was conducted following the same strategy.</span></p>
-<div align="center">
-<table class="MsoTableGrid"
+<div align=center>
-style="border: medium none ; border-collapse: collapse;" border="1"
-cellpadding="0" cellspacing="0">
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-<tbody>
+ style='border-collapse:collapse;border:none'>
-<tr style="height: 129.95pt;">
+ <tr style='height:129.95pt'>
-<td
+  <td width=417 valign=top style='width:312.75pt;border:solid windowtext 1.0pt;
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"
+  padding:0cm 5.4pt 0cm 5.4pt;height:129.95pt'>
-valign="top" width="417">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<p class="MsoNormal"
+cm;line-height:normal'><span lang=EN-US>&nbsp;</span></p>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+  <p class=MsoNormal align=right style='margin-bottom:0cm;margin-bottom:.0001pt;
-lang="EN-US">&nbsp;</span></p>
+  text-align:right;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
-<p class="MsoNormal"
+  width=397 height=194 id="Grafik 77"
-style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+  src="Freiburg10_Modularization_GOI-Dateien/image004.gif"
-align="right"><img id="Grafik 77"
+  alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"></p>
-src="Freiburg10_Modularization_GOI-Dateien/image004.gif"
+  <p class=MsoCaption style='text-indent:0cm'><span lang=EN-US>Figure </span><span lang=EN-US>5</span><span lang=EN-US>: Assembly intermediate in fusion of the
-alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"
+  vectorplasmids containing different promoters. </span><span lang=EN-US
-height="194" width="397"></p>
+  style='font-weight:normal'>Fusion of the BioBrick part <i>beta-globin</i> (</span><span
-<p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure
+  lang=EN-US style='color:black'>BBa_K404107</span><span lang=EN-US
-</span><span lang="EN-US">5</span><span lang="EN-US">: Assembly
+  style='font-weight:normal'>) intron to the composite parts leftITR_pCMV and
-intermediate in fusion of the vectorplasmids containing different
+  leftITR_phTERT, respectively, was performed following the BioBrick assembly
-promoters. </span><span style="font-weight: normal;" lang="EN-US">Fusion
+  strategy by digesting the insert with PstI and XbaI and the vectors with SpeI
-of the BioBrick part <i>beta-globin</i> (</span><span
+  and PstI. The left lane shows the expected fragment at around 560 bp which
-style="color: black;" lang="EN-US">BBa_K404107</span><span
+  corresponds to the <i>beta-globin</i> intron fragment, in contrast to the two
-style="font-weight: normal;" lang="EN-US">) intron to the composite
+  lanes in the center and on the right which correspond to linearized plasmids
-parts leftITR_pCMV and leftITR_phTERT, respectively, was performed
+  after digesting with above mentioned iGEM restriction sites. M, GeneRuler DNA
-following the BioBrick assembly strategy by digesting the insert with
+  ladder mix; Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV,
-PstI and XbaI and the vectors with SpeI and PstI. The left lane shows
+  pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p>
-the expected fragment at around 560 bp which corresponds to the <i>beta-globin</i>
+  </td>
-intron fragment, in contrast to the two lanes in the center and on the
+ </tr>
-right which correspond to linearized plasmids after digesting with
-above mentioned iGEM restriction sites. M, GeneRuler DNA ladder mix;
-Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV,
-pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p>
-</td>
-</tr>
-</tbody>
 </table>
 </div>
-<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<p class="MsoNormal"><span lang="EN-US">Separated fragments were
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-extracted using
-the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated
+<p class=MsoNormal><span lang=EN-US>Separated fragments were extracted using
-with
+the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated with
-T4-ligase. After ligation has been carried out, <i>E. coli</i> XL-1B
+T4-ligase. After ligation has been carried out, <i>E. coli</i> XL-1B cells were
-cells were
 transformed and incubated over night at 37°C. Picking clones from the
 transformation plate was performed the following day and DYT medium was
-inoculated incubating overnight. Plasmid DNA was isolated and test
+inoculated incubating overnight. Plasmid DNA was isolated and test digestion
-digestion
+revealed that cloning was successful obtaining the composite part leftITR_CMV_<i>beta-globin</i>
-revealed that cloning was successful obtaining the composite part
-leftITR_CMV_<i>beta-globin</i>
 intron (BBa_K404117).</span></p>
-<p class="MsoNormal"><span lang="EN-US">Plasmid production
-incorporating all
+<p class=MsoNormal><span lang=EN-US>Plasmid production incorporating all
-required elements for transgene expression and genome encapsidation
+required elements for transgene expression and genome encapsidation into empty
-into empty
+viral capsids was performed by fusing the downstream elements consisting of the
-viral capsids was performed by fusing the downstream elements
+hGH terminator and right ITR to the intermediate part providing the gene of
-consisting of the
-hGH terminator and right ITR to the intermediate part providing the
-gene of
 interest and the promoter fused to the left ITR. </span><span
-lang="EN-US">Figure 5</span><span lang="EN-US"> demonstrates the
+lang=EN-US>Figure 5</span><span lang=EN-US> demonstrates the assembly performed
-assembly performed
 with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus and
-pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after
+pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after digestion on the
-digestion on the
+left lane fits to the hGH-terminator_rightITR length. The isolated fragments
-left lane fits to the hGH-terminator_rightITR length. The isolated
+were ligated and successful assembly was confirmed by test digestion obtaining
-fragments
+the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus_hGH_rightITR
-were ligated and successful assembly was confirmed by test digestion
+(</span><span lang=EN-US style='line-height:200%;color:black'>BBa_K404124</span><span
-obtaining
+lang=EN-US>). </span></p>
-the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i>
-intron_mVenus_hGH_rightITR
+<div align=center>
-(</span><span style="line-height: 200%; color: black;" lang="EN-US">BBa_K404124</span><span
-lang="EN-US">). </span></p>
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-<div align="center">
+ style='border-collapse:collapse;border:none'>
-<table class="MsoTableGrid"
+ <tr style='height:136.9pt'>
-style="border: medium none ; border-collapse: collapse;" border="1"
+  <td width=411 valign=top style='width:308.4pt;border:solid windowtext 1.0pt;
-cellpadding="0" cellspacing="0">
+  padding:0cm 5.4pt 0cm 5.4pt;height:136.9pt'>
-<tbody>
+  <p class=MsoNormal align=right style='margin-bottom:0cm;margin-bottom:.0001pt;
-<tr style="height: 136.9pt;">
+  text-align:right;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
-<td
+  width=397 height=184 id="Grafik 80"
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"
+  src="Freiburg10_Modularization_GOI-Dateien/image005.gif"
-valign="top" width="411">
+  alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"></p>
-<p class="MsoNormal"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784510"><span
-style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+  lang=EN-US>Figure </span></a><span lang=EN-US>6</span><span lang=EN-US
-align="right"><img id="Grafik 80"
+  style='font-weight:normal'>: </span><span lang=EN-US>Modularization of the
-src="Freiburg10_Modularization_GOI-Dateien/image005.gif"
+  assembled vectorplasmid containing the phTERT promoter and mVenus as gene of
-alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"
+  interest.</span><span lang=EN-US style='font-weight:normal'> Fusion of the
-height="184" width="397"></p>
+  composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part  to the
-<p class="MsoCaption" style="text-indent: 0cm;"><a
+  composite parts pSB1C3_hGH_rightITR was performed following the BioBrick
-name="_Ref275784510"><span lang="EN-US">Figure </span></a><span
+  assembly strategy by digesting the insert with XbaI and PstI and the vector
-lang="EN-US">6</span><span style="font-weight: normal;" lang="EN-US">:
+  with SpeI and PstI. The left lane corresponds to linearized plasmid after
-</span><span lang="EN-US">Modularization of the assembled
+  digesting with above mentioned iGEM restriction sites whereas the right lane
-vectorplasmid containing the phTERT promoter and mVenus as gene of
+  reveals an intensive band at around 650 bp confirming the expected size of
-interest.</span><span style="font-weight: normal;" lang="EN-US"> Fusion
+bp of hGH_rITR. M, GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin
-of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part
+  intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p>
-to the composite parts pSB1C3_hGH_rightITR was performed following the
+  </td>
-BioBrick assembly strategy by digesting the insert with XbaI and PstI
+ </tr>
-and the vector with SpeI and PstI. The left lane corresponds to
-linearized plasmid after digesting with above mentioned iGEM
-restriction sites whereas the right lane reveals an intensive band at
-around 650 bp confirming the expected size of 657 bp of hGH_rITR. M,
-GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin
-intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</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">Since cloning does not confirm
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
-biological
+<p class=MsoNormal><span lang=EN-US>Since cloning does not confirm biological
 activity, we analyzed the plasmids and their functional components, hGH
-terminator and <i>beta-globin</i> intron, in cell culture. Assembled
+terminator and <i>beta-globin</i> intron, in cell culture. Assembled plasmids have
-plasmids have
+been cotransfected, using AAV-293 cells, which provide the stable integrated
-been cotransfected, using AAV-293 cells, which provide the stable
+E1A and E1B genes, with helper plasmids required for capsid assembly  and
-integrated
-E1A and E1B genes, with helper plasmids required for capsid assembly
-and
 genome encapsidation (pRC and pHelper) in a molar ratio of 1:1:1
-(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA
+(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA were
-were
+harvested 72-hours post transfection and HT1080 cells transduced with constant
-harvested 72-hours post transfection and HT1080 cells transduced with
+volumes of viral vectors. 48-hours post infection; transduced cells expressing
-constant
-volumes of viral vectors. 48-hours post infection; transduced cells
-expressing
 the gene of interest were analyzed by flow cytometry. Facilitating and
-demonstrating the analysis of functionality of the assembled plasmid,
+demonstrating the analysis of functionality of the assembled plasmid, mVenus
-mVenus
+was used in first place since fluorescent proteins enable facile visualization
-was used in first place since fluorescent proteins enable facile
-visualization
 using fluorescent microscopy and flow cytometry analysis.</span></p>
-<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800683"></a><a
-name="_Toc275797956"><span lang="EN-US"><span
+<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800683"></a><a
-style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
+name="_Toc275797956"><span lang=EN-US><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-lang="EN-US">Testing functionality of Assembled Vectorplasmid</span></a></h3>
+</span></span><span lang=EN-US>Testing functionality of Assembled Vectorplasmid</span></a></h3>
-<h4><a name="_Toc275800684"></a><a name="_Toc275797957"><span
-lang="EN-US">Fluorescence
+<h4><a name="_Toc275800684"></a><a name="_Toc275797957"><span lang=EN-US>Fluorescence
 Microscopy of Target Cells Demonstrates GOI Expression</span></a></h4>
-<p class="MsoNormal"><span lang="EN-US">Qualitative analysis of mVenus
-expression
+<p class=MsoNormal><span lang=EN-US>Qualitative analysis of mVenus expression
-by fluorescence microscopy was conducted using Axio Observer Z1 showing
+by fluorescence microscopy was conducted using Axio Observer Z1 showing that
-that
+transduced HT1080 cells and non-transduced cells could be easily distinguished.
-transduced HT1080 cells and non-transduced cells could be easily
+In </span><span lang=EN-US>Figure 6</span><span lang=EN-US> cells were excited
-distinguished.
+with 505nm and fluorescence emission at 536nm was detected. Therefore, successful
-In </span><span lang="EN-US">Figure 6</span><span lang="EN-US"> cells
+infection of tumor cells by recombinant viral particles carrying the assembled vectorplasmid
-were excited
-with 505nm and fluorescence emission at 536nm was detected. Therefore,
-successful
-infection of tumor cells by recombinant viral particles carrying the
-assembled vectorplasmid
 coding for mVenus could be demonstrated. </span></p>
-<table class="MsoTableGrid"
-style="border: medium none ; border-collapse: collapse;" border="1"
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-cellpadding="0" cellspacing="0">
+ style='border-collapse:collapse;border:none'>
-<tbody>
+ <tr>
-<tr>
+  <td width=334 valign=top style='width:250.7pt;border:solid windowtext 1.0pt;
-<td
+  padding:0cm 5.4pt 0cm 5.4pt'>
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 250.7pt;"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-valign="top" width="334">
+cm;line-height:normal'><span lang=EN-US>A</span></p>
-<p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+  text-align:center;text-indent:0cm;line-height:normal'><img width=264
-lang="EN-US">A</span></p>
+  height=218 id="Grafik 18"
-<p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI-Dateien/image006.jpg"
-style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  alt="Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"></p>
-align="center"><img id="Grafik 18"
+  </td>
-src="Freiburg10_Modularization_GOI-Dateien/image006.jpg"
+  <td width=307 valign=top style='width:230.4pt;border:solid windowtext 1.0pt;
-alt="Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"
+  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-height="218" width="264"></p>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-</td>
+cm;line-height:normal'><span lang=EN-US>B</span></p>
-<td
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><img width=242
-valign="top" width="307">
+  height=220 id="Grafik 19"
-<p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI-Dateien/image007.jpg"
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+  alt="Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"></p>
-lang="EN-US">B</span></p>
+  </td>
-<p class="MsoNormal"
+ </tr>
-style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+ <tr>
-align="center"><img id="Grafik 19"
+  <td width=334 valign=top style='width:250.7pt;border:solid windowtext 1.0pt;
-src="Freiburg10_Modularization_GOI-Dateien/image007.jpg"
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-alt="Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-height="220" width="242"></p>
+cm;line-height:normal'><span lang=EN-US>C</span></p>
-</td>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-</tr>
+  text-align:center;text-indent:0cm;line-height:normal'><img width=258
-<tr>
+  height=195 id="Grafik 16"
-<td
+  src="Freiburg10_Modularization_GOI-Dateien/image008.jpg"
-style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 250.7pt;"
+  alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"></p>
-valign="top" width="334">
+  </td>
-<p class="MsoNormal"
+  <td width=307 valign=top style='width:230.4pt;border-top:none;border-left:
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-lang="EN-US">C</span></p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-<p class="MsoNormal"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+cm;line-height:normal'><span lang=EN-US>D</span></p>
-align="center"><img id="Grafik 16"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-src="Freiburg10_Modularization_GOI-Dateien/image008.jpg"
+  text-align:center;text-indent:0cm;line-height:normal'><img width=257
-alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+  height=194 id="Grafik 17"
-height="195" width="258"></p>
+  src="Freiburg10_Modularization_GOI-Dateien/image009.jpg"
-</td>
+  alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"></p>
-<td
+  </td>
-style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"
+ </tr>
-valign="top" width="307">
+ <tr>
-<p class="MsoNormal"
+  <td width=641 colspan=2 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-lang="EN-US">D</span></p>
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784524"><span
-<p class="MsoNormal"
+  lang=EN-US>Figure </span></a><span lang=EN-US>7</span><span lang=EN-US>: </span><span
-style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  lang=EN-US style='font-weight:normal'>Fluorescence microscopy (Exciatation:
-align="center"><img id="Grafik 17"
+nm, Emission: 536nm) was performed for detection of transduced cell
-src="Freiburg10_Modularization_GOI-Dateien/image009.jpg"
+  expression mVenus. A:Cells detected in bright field picture B: Detection of
-alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+  mVenus expression can be observed.</span></p>
-height="194" width="257"></p>
+  </td>
-</td>
+ </tr>
-</tr>
-<tr>
-<td colspan="2"
-style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
-valign="top" width="641">
-<p class="MsoCaption" style="text-indent: 0cm;"><a
-name="_Ref275784524"><span lang="EN-US">Figure </span></a><span
-lang="EN-US">7</span><span lang="EN-US">: </span><span
-style="font-weight: normal;" lang="EN-US">Fluorescence microscopy
-(Exciatation: 505nm, Emission: 536nm) was performed for detection of
-transduced cell expression mVenus. A:Cells detected in bright field
-picture B: Detection of mVenus expression can be observed.</span></p>
-</td>
-</tr>
-</tbody>
 </table>
-<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<h4><a name="_Toc275800685"></a><a name="_Toc275797958"><span
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-lang="EN-US">Analysis
+<h4><a name="_Toc275800685"></a><a name="_Toc275797958"><span lang=EN-US>Analysis
 of Target Cells by Flow Cytometry demonstrates GOI Expression</span></a></h4>
-<p class="MsoNormal"><span lang="EN-US">Characterizing the function of
-the hGH
+<p class=MsoNormal><span lang=EN-US>Characterizing the function of the hGH
-terminator, the <i>beta-globin</i> intron and the complete plasmid,
+terminator, the <i>beta-globin</i> intron and the complete plasmid, several
-several
 approaches were conducted followed by analysis via flow cytometry. </span></p>
-<h5><a name="_Toc275800686"></a><a name="_Toc275797959"><span
-lang="EN-US">Influence
+<h5><a name="_Toc275800686"></a><a name="_Toc275797959"><span lang=EN-US>Influence
 of hGH terminator BioBrick on GOI Expression</span></a></h5>
-<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg provides
-the hGH
+<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg provides the hGH
-plolyadenylation sequence within the ‘Virus Construction Kit’ due to
+plolyadenylation sequence within the ‘Virus Construction Kit’ due to the fact
-the fact
+that almost every eukaryotic mRNA is processed at their 3´ and 5´end except for
-that almost every eukaryotic mRNA is processed at their 3´ and 5´end
+histone mRNAs </span><span
-except for
+lang=EN-US>(Millevoi et al. 2006)</span><span lang=EN-US>. Pre-mRNAs contain
-histone mRNAs </span><span lang="EN-US">(Millevoi et al. 2006)</span><span
+two canonical conserved sequences. First, the polyadenylation signal “AATAAA”
-lang="EN-US">. Pre-mRNAs contain
+which is recognized by the multiprotein complex and second the GT-rich region
-two canonical conserved sequences. First, the polyadenylation signal
+(downstream sequence element, DSE) which is located 30 nucleotides downstream
-“AATAAA”
+of the cleavage site. The assembled 3´end-processing machinery cleaves the mRNA
-which is recognized by the multiprotein complex and second the GT-rich
+transcript immediately after a CA-nucleotide therefore defining the cleavage
-region
+site </span><span
-(downstream sequence element, DSE) which is located 30 nucleotides
+lang=EN-US>(Danckwardt et al. 2008)</span><span lang=EN-US style='font-size:
-downstream
+.0pt;line-height:200%'>. </span><span lang=EN-US>Recombinant vectorplasmids
-of the cleavage site. The assembled 3´end-processing machinery cleaves
+were engineered containing the inverted terminal repeats (ITRs), a strong
-the mRNA
+eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of interest
-transcript immediately after a CA-nucleotide therefore defining the
+with and without the hGH terminator signal. Transduction of HT1080 cells with constant
-cleavage
+volume of viral particles containing the vectorplasmids and measuring mVenus
-site </span><span lang="EN-US">(Danckwardt et al. 2008)</span><span
-style="font-size: 12pt; line-height: 200%;" lang="EN-US">. </span><span
-lang="EN-US">Recombinant vectorplasmids
-were engineered containing the inverted terminal repeats (ITRs), a
-strong
-eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of
-interest
-with and without the hGH terminator signal. Transduction of HT1080
-cells with constant
-volume of viral particles containing the vectorplasmids and measuring
-mVenus
 expression 24-hours post infection by flow cytometry demonstrated that
-transgene expression of the constructs lacking the hGH termination
+transgene expression of the constructs lacking the hGH termination signal is
-signal is
+significantly reduced as shown in </span><span
-significantly reduced as shown in </span><span lang="EN-US">Figure 7</span><span
+lang=EN-US>Figure 7</span><span lang=EN-US> and </span><span
-lang="EN-US"> and </span><span lang="EN-US">Figure 8</span><span
+lang=EN-US>Figure 8</span><span lang=EN-US> confirming the expected results
-lang="EN-US"> confirming the expected results
+that hGH is essential for mRNA processing. The iGEM team Freiburg_Bioware 2010
-that hGH is essential for mRNA processing. The iGEM team
+therefore suggests using the provided hGH termination signal within the Virus
-Freiburg_Bioware 2010
-therefore suggests using the provided hGH termination signal within the
-Virus
 Construction Kit for optimal gene expression.</span></p>
-<table class="MsoTableGrid"
-style="border: medium none ; border-collapse: collapse;" border="1"
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-cellpadding="0" cellspacing="0">
+ style='border-collapse:collapse;border:none'>
-<tbody>
+ <tr>
-<tr>
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-<td
+  padding:0cm 5.4pt 0cm 5.4pt'>
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-valign="top" width="641">
+cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid lacking hGH
-<p class="MsoNormal"
+  termination signal</span></b></p>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-lang="EN-US">Vectorplasmid lacking hGH termination signal</span></b></p>
+cm;line-height:normal'><img width=629 height=408 id="Grafik 30"
-<p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI-Dateien/image010.gif"></p>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
+  </td>
-id="Grafik 30" src="Freiburg10_Modularization_GOI-Dateien/image010.gif"
+ </tr>
-height="408" width="629"></p>
+ <tr>
-</td>
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-</tr>
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-<tr>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<td
+cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid containing hGH
-style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+  terminator signal</span></b></p>
-valign="top" width="641">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<p class="MsoNormal"
+cm;line-height:normal;page-break-after:avoid'><img width=635 height=410
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
+  id="Grafik 2049" src="Freiburg10_Modularization_GOI-Dateien/image011.gif"></p>
-lang="EN-US">Vectorplasmid containing hGH terminator signal</span></b></p>
+  </td>
-<p class="MsoNormal"
+ </tr>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+ <tr>
-id="Grafik 2049"
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-src="Freiburg10_Modularization_GOI-Dateien/image011.gif" height="410"
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-width="635"></p>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-</td>
+cm;line-height:normal'><a name="_Ref275784539"><b><span lang=EN-US>Figure </span></b></a><b><span lang=EN-US>8</span></b><b><span lang=EN-US>:</span></b><span lang=EN-US> <b>Flow
-</tr>
+  cytometry analysis of vectorplasmids with and without hGH terminator.</b> A:
-<tr>
+  Gating non transduced cells (control); subcellular debris and clumps can be
-<td
+  distinguished from single cells by size, estimated forward scatter (FS Lin)
-style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+  and granularity, estimated side scatter (SS Lin) B: Non transduced cells
-valign="top" width="641">
+  applied against mVenus (Analytical gate was set such that 1% or fewer of
-<p class="MsoNormal"
+  negative control cells fell within the positive region (R5). C: Gating transduced
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a
+  cells (R2 </span><span lang=EN-US style='font-family:"Cambria Math","serif"'>&#8793;</span><span
-name="_Ref275784539"><b><span lang="EN-US">Figure </span></b></a><b><span
+  lang=EN-US>R14) (used plasmids for transfection: GOI:
-lang="EN-US">8</span></b><b><span lang="EN-US">:</span></b><span
+  pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR (BBa_K404127), pHelper, pRC).
-lang="EN-US"> <b>Flow cytometry analysis of vectorplasmids with and
+  D: Transduced cells plotted against mVenus, R10 comprises transduced cells by
-without hGH terminator.</b> A: Gating non transduced cells (control);
+  detecting mVenus expression. E: Overlay of non-transduced (red) and
-subcellular debris and clumps can be distinguished from single cells by
+  transduced (green) cells applied against mVenus.F: Gating non-transduced
-size, estimated forward scatter (FS Lin) and granularity, estimated
+  cells (control) G: Non-transduced cells applied against mVenus. H: Gating
-side scatter (SS Lin) B: Non transduced cells applied against mVenus
+  transduced cells (R2 </span><span lang=EN-US style='font-family:"Cambria Math","serif"'>&#8793;</span><span
-(Analytical gate was set such that 1% or fewer of negative control
+  lang=EN-US>R14) (used plasmids for transfection: GOI: reassembled pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-cells fell within the positive region (R5). C: Gating transduced cells
+  (BBa_K404119), pHelper, pRC). I: Transduced cells applied against mVenus, R10
-(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
+  comprised transduced cells, by detecting mVenus expression. J: Overlay of
-lang="EN-US">≙</span><span lang="EN-US">R14) (used plasmids for
+  non-transduced (red) and transduced (green) cells applied against mVenus.</span></p>
-transfection: GOI: pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR
+  </td>
-(BBa_K404127), pHelper, pRC). D: Transduced cells plotted against
+ </tr>
-mVenus, R10 comprises transduced cells by detecting mVenus expression.
-E: Overlay of non-transduced (red) and transduced (green) cells applied
-against mVenus.F: Gating non-transduced cells (control) G:
-Non-transduced cells applied against mVenus. H: Gating transduced cells
-(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
-lang="EN-US">≙</span><span lang="EN-US">R14) (used plasmids for
-transfection: GOI: reassembled
-pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119), pHelper,
-pRC). I: Transduced cells applied against mVenus, R10 comprised
-transduced cells, by detecting mVenus expression. J: Overlay of
-non-transduced (red) and transduced (green) cells applied against
-mVenus.</span></p>
-</td>
-</tr>
-</tbody>
 </table>
-<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<table class="MsoTableGrid"
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-style="border: medium none ; border-collapse: collapse;" border="1"
-cellpadding="0" cellspacing="0">
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-<tbody>
+ style='border-collapse:collapse;border:none'>
-<tr>
+ <tr>
-<td
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+  padding:0cm 5.4pt 0cm 5.4pt'>
-valign="top" width="641">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<p class="MsoNormal"
+cm;line-height:normal'><span lang=EN-US>&nbsp;</span></p>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-lang="EN-US">&nbsp;</span></p>
+cm;line-height:normal;page-break-after:avoid'><span lang=EN-US>                          </span><img
-<p class="MsoNormal"
+  width=473 height=355 id="Diagramm 3"
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><span
+  src="Freiburg10_Modularization_GOI-Dateien/image012.gif"></p>
-lang="EN-US"> </span><img id="Diagramm 3"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784545"><span
-src="Freiburg10_Modularization_GOI-Dateien/image012.gif" height="355"
+  lang=EN-US>Figure </span></a><span lang=EN-US>9</span><span lang=EN-US>: Flow
-width="473"></p>
+  cytometry analysis of vectorplasmids with and without hGH terminator.</span><span
-<p class="MsoCaption" style="text-indent: 0cm;"><a
+  lang=EN-US style='font-weight:normal'> YFP expression of viral genomes was
-name="_Ref275784545"><span lang="EN-US">Figure </span></a><span
+  determined by flow cytomery after 24-hour post infection. Results demonstrate
-lang="EN-US">9</span><span lang="EN-US">: Flow cytometry analysis of
+  that mVenus expression of vectorplasmids lacking the hGH terminator is
-vectorplasmids with and without hGH terminator.</span><span
+  reduced significantly proving that the polyadenylation signal is essential
-style="font-weight: normal;" lang="EN-US"> YFP expression of viral
+  for viral gene expression using recombinant viral vectors engineered by using
-genomes was determined by flow cytomery after 24-hour post infection.
+  components of the Virus Construction Kit.</span></p>
-Results demonstrate that mVenus expression of vectorplasmids lacking
+  </td>
-the hGH terminator is reduced significantly proving that the
+ </tr>
-polyadenylation signal is essential for viral gene expression using
-recombinant viral vectors engineered by using components of the Virus
-Construction Kit.</span></p>
-</td>
-</tr>
-</tbody>
 </table>
-<p class="MsoCaption"><span lang="EN-US">&nbsp;</span></p>
-<h5><a name="_Toc275800687"></a><a name="_Toc275797960"><span
+<p class=MsoCaption><span lang=EN-US>&nbsp;</span></p>
-lang="EN-US">Influence
+<h5><a name="_Toc275800687"></a><a name="_Toc275797960"><span lang=EN-US>Influence
 of <i>Beta-globin</i> intron Biobrick on GOI Expression</span></a></h5>
-<p class="MsoNormal"><span lang="EN-US">Providing an element assumed to
-be an
+<p class=MsoNormal><span lang=EN-US>Providing an element assumed to be an
-enhancer of transgene expression </span><span lang="EN-US">(Nott et
+enhancer of transgene expression </span><span
-al. 2003)</span><span lang="EN-US">, the iGEM team Freiburg tested
+lang=EN-US>(Nott et al. 2003)</span><span lang=EN-US>, the iGEM team Freiburg tested
-a beta-globin intron derived from the human <i>beta globin</i> gene
+a beta-globin intron derived from the human <i>beta globin</i> gene which can
-which can
+be fused upstream of the desired gene of interest. The beta-globin intron
-be fused upstream of the desired gene of interest. The beta-globin
+BioBrick consists of a partial chimeric CMV promoter followed by the intron II
-intron
+of the <i>beta-globin</i> gene. The 3´end of the intron is fused to the first 25
-BioBrick consists of a partial chimeric CMV promoter followed by the
+bases of human <i>beta globin</i> gene exon 3. The <i>beta globin</i> intron
-intron II
+BioBrick is assumed to enhance eukaryotic gene expression </span><span lang=EN-US>(Nott et al. 2003)</span><span lang=EN-US>. Analysis was conducted as
-of the <i>beta-globin</i> gene. The 3´end of the intron is fused to
+described for the hGH terminator experiment (see above). As shown in </span><span lang=EN-US>Figure 9</span><span lang=EN-US> and </span><span
-the first 25
+lang=EN-US>Figure 10</span><span lang=EN-US> the vectorplasmid missing the <i>beta-globin</i>
-bases of human <i>beta globin</i> gene exon 3. The <i>beta globin</i>
+intron showed a negligible difference in mVenus expression compared to viral
-intron
+genomes containing the <i>beta-globin</i> intron. Considering these results and
-BioBrick is assumed to enhance eukaryotic gene expression </span><span
+taking into account that a constant volume of viral particles has been used for
-lang="EN-US">(Nott et al. 2003)</span><span lang="EN-US">. Analysis
+transduction, the difference between the construct containing and lacking the
-was conducted as
-described for the hGH terminator experiment (see above). As shown in </span><span
-lang="EN-US">Figure 9</span><span lang="EN-US"> and </span><span
-lang="EN-US">Figure 10</span><span lang="EN-US"> the vectorplasmid
-missing the <i>beta-globin</i>
-intron showed a negligible difference in mVenus expression compared to
-viral
-genomes containing the <i>beta-globin</i> 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 </span><span lang="EN-US">(Dong
+decreases with increasing sizes of the insert </span><span
-et al. 1996)</span><span lang="EN-US">, the iGEM team
+lang=EN-US>(Dong et al. 1996)</span><span lang=EN-US>, the iGEM team
-Freiburg_Bioware suggests using the <i>beta-globin </i>intron in
+Freiburg_Bioware suggests using the <i>beta-globin </i>intron in dependence on
-dependence on
 the size of your transgene.</span></p>
-<table class="MsoTableGrid"
-style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 width=654
-border="1" cellpadding="0" cellspacing="0" width="654">
+ style='width:490.75pt;border-collapse:collapse;border:none'>
-<tbody>
+ <tr style='height:2.5pt'>
-<tr style="height: 2.5pt;">
+  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
-<td
+  padding:0cm 5.4pt 0cm 5.4pt;height:2.5pt'>
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-valign="top" width="654">
+cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid lacking <i>beta-globin</i>
-<p class="MsoNormal"
+  intron</span></b></p>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-lang="EN-US">Vectorplasmid lacking <i>beta-globin</i> intron</span></b></p>
+cm;line-height:normal'><img width=640 height=412 id="Grafik 55"
-<p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI-Dateien/image013.gif"></p>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
+  </td>
-id="Grafik 55" src="Freiburg10_Modularization_GOI-Dateien/image013.gif"
+ </tr>
-height="412" width="640"></p>
+ <tr style='height:106.5pt'>
-</td>
+  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
-</tr>
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:106.5pt'>
-<tr style="height: 106.5pt;">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<td
+cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid containing <i>beta-globin</i>
-style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
+  intron</span></b></p>
-valign="top" width="654">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<p class="MsoNormal"
+cm;line-height:normal;page-break-after:avoid'><img width=635 height=410
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
+  id="Grafik 63" src="Freiburg10_Modularization_GOI-Dateien/image014.gif"></p>
-lang="EN-US">Vectorplasmid containing <i>beta-globin</i> intron</span></b></p>
+  </td>
-<p class="MsoNormal"
+ </tr>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+ <tr style='height:106.5pt'>
-id="Grafik 63" src="Freiburg10_Modularization_GOI-Dateien/image014.gif"
+  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
-height="410" width="635"></p>
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:106.5pt'>
-</td>
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784803"><span
-</tr>
+  lang=EN-US>Figure </span></a><span lang=EN-US>10</span><span lang=EN-US>: Flow
-<tr style="height: 106.5pt;">
+  cytometry analysis of vectorplasmids with and without <i>beta-globin</i>
-<td
+  intron.  A</span><span lang=EN-US style='font-weight:normal'>: Gating non
-style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
+  transduced cells (control); subcellular debris and clumps can be
-valign="top" width="654">
+  distinguished from single cells by size, estimated forward scatter (FS Lin)
-<p class="MsoCaption" style="text-indent: 0cm;"><a
+  and granularity, estimated side scatter (SS Lin) </span><span lang=EN-US>B</span><span
-name="_Ref275784803"><span lang="EN-US">Figure </span></a><span
+  lang=EN-US style='font-weight:normal'>: Non transduced cells applied against
-lang="EN-US">10</span><span lang="EN-US">: Flow cytometry analysis of
+  mVenus (Analytical gate was set such that 1% or fewer of negative control
-vectorplasmids with and without <i>beta-globin</i> intron. A</span><span
+  cells fell within the positive region (R5). </span><span lang=EN-US>C</span><span
-style="font-weight: normal;" lang="EN-US">: Gating non transduced
+  lang=EN-US style='font-weight:normal'>: Gating transduced cells (R2 </span><span
-cells (control); subcellular debris and clumps can be distinguished
+  lang=EN-US style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
-from single cells by size, estimated forward scatter (FS Lin) and
+  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
-granularity, estimated side scatter (SS Lin) </span><span lang="EN-US">B</span><span
+  GOI: </span><span lang=EN-US>pSB1C3_lITR_CMV_mVenus_hGH_rITR (BBa_K404128)</span><span
-style="font-weight: normal;" lang="EN-US">: Non transduced cells
+  lang=EN-US style='font-weight:normal'>, pHelper, pRC). </span><span
-applied against mVenus (Analytical gate was set such that 1% or fewer
+  lang=EN-US>D</span><span lang=EN-US style='font-weight:normal'>: Transduced
-of negative control cells fell within the positive region (R5). </span><span
+  cells plotted against mVenus, R10 comprised transduced cells, by detecting
-lang="EN-US">C</span><span style="font-weight: normal;" lang="EN-US">:
+  mVenus expression </span><span lang=EN-US>E</span><span lang=EN-US
-Gating transduced cells (R2 </span><span
+  style='font-weight:normal'>: Overlay of non-transduced (red) and transduced
-style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
+  (green) cells applied against mVenus </span><span lang=EN-US>F</span><span
-lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
+  lang=EN-US style='font-weight:normal'>: Gating non-transduced cells (control).
-(used plasmids for transfection: GOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_mVenus_hGH_rITR
+  </span><span lang=EN-US>G</span><span lang=EN-US style='font-weight:normal'>:
-(BBa_K404128)</span><span style="font-weight: normal;" lang="EN-US">,
+  Non-transduced cells applied against mVenus (R5).</span><span lang=EN-US>H</span><span
-pHelper, pRC). </span><span lang="EN-US">D</span><span
+  lang=EN-US style='font-weight:normal'>: Gating transduced cells (R2 </span><span
-style="font-weight: normal;" lang="EN-US">: Transduced cells plotted
+  lang=EN-US style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
-against mVenus, R10 comprised transduced cells, by detecting mVenus
+  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
-expression </span><span lang="EN-US">E</span><span
+  GOI: reassembled </span><span lang=EN-US>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
+  (BBa_K404119)</span><span lang=EN-US style='font-weight:normal'>, pHelper, pRC).
-(red) and transduced (green) cells applied against mVenus </span><span
+  </span><span lang=EN-US>I</span><span lang=EN-US style='font-weight:normal'>:
-lang="EN-US">F</span><span style="font-weight: normal;" lang="EN-US">:
+  Transduced cells applied against mVenus, R10 comprised transduced cells, by
-Gating non-transduced cells (control). </span><span lang="EN-US">G</span><span
+  detecting mVenus expression. </span><span lang=EN-US>J</span><span
-style="font-weight: normal;" lang="EN-US">: Non-transduced cells
+  lang=EN-US style='font-weight:normal'>: Overlay of non-transduced (red) and
-applied against mVenus (R5).</span><span lang="EN-US">H</span><span
+  transduced (green) cells applied against mVenus.</span></p>
-style="font-weight: normal;" lang="EN-US">: Gating transduced cells
+  </td>
-(R2 </span><span
+ </tr>
-style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
-lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
-(used plasmids for transfection: GOI: reassembled </span><span
-lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119)</span><span
-style="font-weight: normal;" lang="EN-US">, pHelper, pRC). </span><span
-lang="EN-US">I</span><span style="font-weight: normal;" lang="EN-US">:
-Transduced cells applied against mVenus, R10 comprised transduced
-cells, by detecting mVenus expression. </span><span lang="EN-US">J</span><span
-style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
-(red) and transduced (green) cells applied against mVenus.</span></p>
-</td>
-</tr>
-</tbody>
 </table>
-<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<table class="MsoTableGrid"
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
-border="1" cellpadding="0" cellspacing="0" width="654">
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 width=654
-<tbody>
+ style='width:490.75pt;border-collapse:collapse;border:none'>
-<tr style="height: 90.2pt;">
+ <tr style='height:90.2pt'>
-<td
+  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 90.2pt;"
+  padding:0cm 5.4pt 0cm 5.4pt;height:90.2pt'>
-valign="top" width="654">
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-<p class="MsoNormal"
+  text-align:center;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
-style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+  width=450 height=332 id="Diagramm 57"
-align="center"><img id="Diagramm 57"
+  src="Freiburg10_Modularization_GOI-Dateien/image015.gif"></p>
-src="Freiburg10_Modularization_GOI-Dateien/image015.gif" height="332"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784805"><span
-width="450"></p>
+  lang=EN-US>Figure </span></a><span lang=EN-US>11</span><span lang=EN-US>:
-<p class="MsoCaption" style="text-indent: 0cm;"><a
+  Flow cytometry analysis of vectorplasmids with and without <i>beta-globin</i>
-name="_Ref275784805"><span lang="EN-US">Figure </span></a><span
+  intron.</span><span lang=EN-US style='font-weight:normal'> 48-hours post
-lang="EN-US">11</span><span lang="EN-US">: Flow cytometry analysis of
+  transfection, viral particles were harvested by freeze-thaw lysis and
-vectorplasmids with and without <i>beta-globin</i> intron.</span><span
+  centrifugation followed by HT1080 transduction. YFP expression of vectorplasmids
-style="font-weight: normal;" lang="EN-US"> 48-hours post transfection,
+  was determined by flow cytometry 24-hours post infection. The vectorplasmid
-viral particles were harvested by freeze-thaw lysis and centrifugation
+  missing the beta-globin intron showed a negligible difference in mVenus
-followed by HT1080 transduction. YFP expression of vectorplasmids was
+  expression compared to viral plasmid containing the beta-globin intron.</span></p>
-determined by flow cytometry 24-hours post infection. The vectorplasmid
+  </td>
-missing the beta-globin intron showed a negligible difference in mVenus
+ </tr>
-expression compared to viral plasmid containing the beta-globin intron.</span></p>
-</td>
-</tr>
-</tbody>
 </table>
-<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<h5><a name="_Toc275800688"></a><a name="_Toc275797961"><span
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
-lang="EN-US">Functionality
+<h5><a name="_Toc275800688"></a><a name="_Toc275797961"><span lang=EN-US>Functionality
 of the Full Assembled Vectorplasmid Demonstrated by GOI Expression</span></a><span
-lang="EN-US"> </span></h5>
+lang=EN-US> </span></h5>
-<p class="MsoNormal"><span lang="EN-US">After assembly of plasmids
-containing all
+<p class=MsoNormal><span lang=EN-US>After assembly of plasmids containing all
-required elements (see </span><span lang="EN-US">Figure 1</span><span
+required elements (see </span><span lang=EN-US>Figure 1</span><span lang=EN-US>),
-lang="EN-US">),
+functionality was tested in cell culture. AAV-293 cells stably expressing E1A
-functionality was tested in cell culture. AAV-293 cells stably
+and E1B proteins were transfected with three plasmids  (pHelper, pRC, pGOI).
-expressing E1A
+Virus particles were harvested 72-hours post-transfection and the tumor cell
-and E1B proteins were transfected with three plasmids (pHelper, pRC,
+line HT1080 was transduced with the recombinant viral vectors encapsidating the
-pGOI).
-Virus particles were harvested 72-hours post-transfection and the tumor
-cell
-line HT1080 was transduced with the recombinant viral vectors
-encapsidating the
 gene of interest mVenus (BBa_I757008).</span></p>
-<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware
+<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware 2010
-compared the standard-plasmid containing a subcloned mVenus
+compared the standard-plasmid containing a subcloned mVenus (pAAV_mVenus,
-(pAAV_mVenus,
+derived from the Stratagene system) with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-derived from the Stratagene system) with the assembled plasmid
+(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by flow
-pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+cytometry analysis are shown in </span><span lang=EN-US>Figure 11</span><span
-(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by
+lang=EN-US> and </span><span lang=EN-US>Figure 12</span><span lang=EN-US>.
-flow
+Comparing mVenus expression of the standard plasmid and the modified, assembled
-cytometry analysis are shown in </span><span lang="EN-US">Figure 11</span><span
+plasmid reveals that biological functionality of the reassembled plasmid was
-lang="EN-US"> and </span><span lang="EN-US">Figure 12</span><span
-lang="EN-US">.
-Comparing mVenus expression of the standard plasmid and the modified,
-assembled
-plasmid reveals that biological functionality of the reassembled
-plasmid was
 confirmed. </span></p>
-<table class="MsoTableGrid"
-style="border: medium none ; border-collapse: collapse;" border="1"
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-cellpadding="0" cellspacing="0">
+ style='border-collapse:collapse;border:none'>
-<tbody>
+ <tr>
-<tr>
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-<td
+  padding:0cm 5.4pt 0cm 5.4pt'>
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-valign="top" width="641">
+cm;line-height:normal'><b>pSB1C3_mVenus (BBa_K404119)</b></p>
-<p class="MsoNormal"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>pSB1C3_mVenus
+cm;line-height:normal'><img width=634 height=409 id="Grafik 2068"
-(BBa_K404119)</b></p>
+  src="Freiburg10_Modularization_GOI-Dateien/image016.gif"></p>
-<p class="MsoNormal"
+  </td>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
+ </tr>
-id="Grafik 2068"
+ <tr>
-src="Freiburg10_Modularization_GOI-Dateien/image016.gif" height="409"
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-width="634"></p>
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-</td>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-</tr>
+cm;line-height:normal'><b><span lang=EN-US>pAAV_mVenus (Stratagene)</span></b></p>
-<tr>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-<td
+cm;line-height:normal;page-break-after:avoid'><img width=630 height=407
-style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+  id="Grafik 89" src="Freiburg10_Modularization_GOI-Dateien/image017.gif"></p>
-valign="top" width="641">
+  </td>
-<p class="MsoNormal"
+ </tr>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
+ <tr>
-lang="EN-US">pAAV_mVenus (Stratagene)</span></b></p>
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-<p class="MsoNormal"
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784576"><span
-id="Grafik 89" src="Freiburg10_Modularization_GOI-Dateien/image017.gif"
+  lang=EN-US>Figure </span></a><span lang=EN-US>12</span><span lang=EN-US>: Flow
-height="407" width="630"></p>
+  cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to
-</td>
+  standard plasmid provided by Stratagene. A</span><span lang=EN-US
-</tr>
+  style='font-weight:normal'>: Gating non transduced cells (control);
-<tr>
+  subcellular debris and clumps can be distinguished from single cells by size,
-<td
+  estimated forward scatter (FS Lin) and granularity, estimated side scatter
-style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+  (SS Lin) B: Non transduced cells plotted against mVenus (Analytical gate was
-valign="top" width="641">
+  set such that 1% or fewer of negative control cells fell within the positive
-<p class="MsoCaption" style="text-indent: 0cm;"><a
+  region (R5).C: Gating transduced cells (R2 </span><span lang=EN-US
-name="_Ref275784576"><span lang="EN-US">Figure </span></a><span
+  style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
-lang="EN-US">12</span><span lang="EN-US">: Flow cytometry analysis of
+  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
-reassembled vectorplasmid (BBa_K404119) compared to standard plasmid
+  pGOI: </span><span lang=EN-US>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-provided by Stratagene. A</span><span style="font-weight: normal;"
+  (pSB1C3_mVenus: </span><span lang=EN-US>BBa_K404119</span><span lang=EN-US
-lang="EN-US">: Gating non transduced cells (control); subcellular
+  style='color:#00B050;font-weight:normal'>)</span><span lang=EN-US
-debris and clumps can be distinguished from single cells by size,
+  style='font-weight:normal'>, pHelper, pRC. </span><span lang=EN-US>D</span><span
-estimated forward scatter (FS Lin) and granularity, estimated side
+  lang=EN-US style='font-weight:normal'>: Transduced cells plotted against
-scatter (SS Lin) B: Non transduced cells plotted against mVenus
+  mVenus, R10 comprised transduced cells, by detecting mVenus expression. </span><span
-(Analytical gate was set such that 1% or fewer of negative control
+  lang=EN-US>E</span><span lang=EN-US style='font-weight:normal'>: Overlay of
-cells fell within the positive region (R5).C: Gating transduced cells
+  non-transduced (red) and transduced (green). </span><span lang=EN-US>F</span><span
-(R2 </span><span
+  lang=EN-US style='font-weight:normal'>: Gating non transduced cells
-style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
+  (control). </span><span lang=EN-US>G</span><span lang=EN-US style='font-weight:
-lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
+  normal'>: Non-transduced cells plotted against mVenus (R5). </span><span
-(used plasmids for transfection: pGOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+  lang=EN-US>H</span><span lang=EN-US style='font-weight:normal'>: Gating
-(pSB1C3_mVenus: </span><span lang="EN-US">BBa_K404119</span><span
+  transduced cells (R14 </span><span lang=EN-US style='font-family:"Cambria Math","serif";
-style="color: rgb(0, 176, 80); font-weight: normal;" lang="EN-US">)</span><span
+  font-weight:normal'>&#8793;</span><span lang=EN-US style='font-weight:normal'>R2)
-style="font-weight: normal;" lang="EN-US">, pHelper, pRC. </span><span
+  (used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). </span><span
-lang="EN-US">D</span><span style="font-weight: normal;" lang="EN-US">:
+  lang=EN-US>I</span><span lang=EN-US style='font-weight:normal'>: Transduced
-Transduced cells plotted against mVenus, R10 comprised transduced
+  cells plotted against mVenus, R10 comprised transduced cells, by detecting
-cells, by detecting mVenus expression. </span><span lang="EN-US">E</span><span
+  mVenus expression.</span><span lang=EN-US> J</span><span lang=EN-US
-style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
+  style='font-weight:normal'>: Overlay of non-transduced (red) and transduced
-(red) and transduced (green). </span><span lang="EN-US">F</span><span
+  (green) cells plotted against mVenus expression. </span></p>
-style="font-weight: normal;" lang="EN-US">: Gating non transduced
+  </td>
-cells (control). </span><span lang="EN-US">G</span><span
+ </tr>
-style="font-weight: normal;" lang="EN-US">: Non-transduced cells
-plotted against mVenus (R5). </span><span lang="EN-US">H</span><span
-style="font-weight: normal;" lang="EN-US">: Gating transduced cells
-(R14 </span><span
-style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
-lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R2)
-(used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). </span><span
-lang="EN-US">I</span><span style="font-weight: normal;" lang="EN-US">:
-Transduced cells plotted against mVenus, R10 comprised transduced
-cells, by detecting mVenus expression.</span><span lang="EN-US"> J</span><span
-style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
-(red) and transduced (green) cells plotted against mVenus expression. </span></p>
-</td>
-</tr>
-</tbody>
 </table>
-<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
-<table class="MsoTableGrid"
-style="border: medium none ; border-collapse: collapse;" border="1"
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-cellpadding="0" cellspacing="0">
-<tbody>
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-<tr>
+ style='border-collapse:collapse;border:none'>
-<td
+ <tr>
-style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-valign="top" width="641">
+  padding:0cm 5.4pt 0cm 5.4pt'>
-<p class="MsoNormal"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+cm;line-height:normal'><span lang=EN-US>&nbsp;</span></p>
-lang="EN-US">&nbsp;</span></p>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-<p class="MsoNormal"
+  text-align:center;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
-style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+  width=541 height=396 id="Diagramm 90"
-align="center"><img id="Diagramm 90"
+  src="Freiburg10_Modularization_GOI-Dateien/image018.gif"></p>
-src="Freiburg10_Modularization_GOI-Dateien/image018.gif" height="396"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784852"><span
-width="541"></p>
+  lang=EN-US>Figure </span></a><span lang=EN-US>13</span><span lang=EN-US>:
-<p class="MsoCaption" style="text-indent: 0cm;"><a
+  Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared
-name="_Ref275784852"><span lang="EN-US">Figure </span></a><span
+  to standard plasmid provided by Stratagene. </span><span lang=EN-US
-lang="EN-US">13</span><span lang="EN-US">: Flow cytometry analysis of
+  style='font-weight:normal'>Fluorescence of the standard plasmid pAAV_mVenus (Stratagene)
-reassembled vectorplasmid (BBa_K404119) compared to standard plasmid
+  and the recombinant pSB1C3_mVenus (BBa_K404119) construct was measured. As
-provided by Stratagene. </span><span style="font-weight: normal;"
+  demonstrated mVenus expression is enhanced in the assembled plasmid
-lang="EN-US">Fluorescence of the standard plasmid pAAV_mVenus
+  (pSB1C3_mVenus) compared to the standard pAAV_mVenus construct.</span></p>
-(Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct
+  </td>
-was measured. As demonstrated mVenus expression is enhanced in the
+ </tr>
-assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus
-construct.</span></p>
-</td>
-</tr>
-</tbody>
 </table>
-<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800689"><span
-lang="EN-US"><span
+<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800689"><span
-style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
+lang=EN-US><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-lang="EN-US">Conclusion</span></a></h3>
+</span></span><span lang=EN-US>Conclusion</span></a></h3>
-<p class="MsoNormal"><span lang="EN-US">Idea of the modular ‘Virus
-Construction
+<p class=MsoNormal><span lang=EN-US>Idea of the modular ‘Virus Construction
-Kit’ is to provide all required elements for producing recombinant,
+Kit’ is to provide all required elements for producing recombinant, functional
-functional
+virus particles delivering encapsidated genes of interest to specific cells.
-virus particles delivering encapsidated genes of interest to specific
+First step was  to modify and modularize the vectorplasmid comprising basically
-cells.
+the cis-elements for replication (ITRs), a strong eukaryotic or tissue specific
-First step was to modify and modularize the vectorplasmid comprising
+promoter (pCMV or phTERT), the gene of interest (fluorescent proteins or
-basically
+suicide genes) and the hGH termination signal. Each element was successfully
-the cis-elements for replication (ITRs), a strong eukaryotic or tissue
+cloned and reassembled resulting in functional vectorplasmids determined by flow
-specific
+cytometry and fluorescence microscopy analyses. Experiments have been performed
-promoter (pCMV or phTERT), the gene of interest (fluorescent proteins
+with mVenus since measurement of fluorescent proteins can be easily performed
-or
+and visualized. Considering the results, the iGEM team Freiburg_Bioware 2010
-suicide genes) and the hGH termination signal. Each element was
+then tested the construct containing the suicide genes thymidine kinase and
-successfully
+cytosine deaminase. Further details demonstrating efficient tumor killing,
-cloned and reassembled resulting in functional vectorplasmids
+using prodrug-activating systems, see results page ‘Arming – Killing the
-determined by flow
-cytometry and fluorescence microscopy analyses. Experiments have been
-performed
-with mVenus since measurement of fluorescent proteins can be easily
-performed
-and visualized. Considering the results, the iGEM team Freiburg_Bioware
-then tested the construct containing the suicide genes thymidine kinase
-and
-cytosine deaminase. Further details demonstrating efficient tumor
-killing,
-using prodrug-activating systems, see results page ‘Arming – Killing
-the
 tumor’. </span></p>
-<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800690"><span
-lang="EN-US"><span
+<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800690"><span
-style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
+lang=EN-US><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-lang="EN-US">References</span></a></h3>
+</span></span><span lang=EN-US>References</span></a></h3>
-<p style="text-indent: 36pt;"><span
-style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
-lang="EN-US">Danckwardt, S., Hentze, M.W. &amp; Kulozik,
+font-family:"Calibri","sans-serif"'>Danckwardt, S., Hentze, M.W. &amp; Kulozik,
-A.E., 2008. 3' end mRNA processing: molecular mechanisms and
+A.E., 2008. 3' end mRNA processing: molecular mechanisms and implications for
-implications for
+health and disease. <i>The EMBO journal</i>, 27(3), 482-98. Available at:
-health and disease. <i>The EMBO journal</i>, 27(3), 482-98. Available
-at:
 http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p>
-<p style="text-indent: 36pt;"><span
-style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
-lang="EN-US">Dong, J.Y., Fan, P.D. &amp; Frizzell, R.a.,
+font-family:"Calibri","sans-serif"'>Dong, J.Y., Fan, P.D. &amp; Frizzell, R.a.,
 . Quantitative analysis of the packaging capacity of recombinant
-adeno-associated virus. <i>Human gene therapy</i>, 7(17), 2101-12.
+adeno-associated virus. <i>Human gene therapy</i>, 7(17), 2101-12. Available
-Available
 at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
-<p style="text-indent: 36pt;"><span
-style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
-lang="EN-US">Millevoi, S. et al., 2006. An interaction
+font-family:"Calibri","sans-serif"'>Millevoi, S. et al., 2006. An interaction
 between U2AF 65 and CF I(m) links the splicing and 3' end processing
 machineries. <i>The EMBO journal</i>, 25(20), 4854-64. Available at:
 http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p>
-<p style="text-indent: 36pt;"><span
-style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
-lang="EN-US">Nott, A., Meislin, S.H. &amp; Moore, M.J.,
+font-family:"Calibri","sans-serif"'>Nott, A., Meislin, S.H. &amp; Moore, M.J.,
-. A quantitative analysis of intron effects on mammalian gene
+. A quantitative analysis of intron effects on mammalian gene expression. <i>RNA
-expression. <i>RNA
 (New York, N.Y.)</i>, 9(5), 607-17. Available at:
 http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
-<p style="text-indent: 36pt;"><span lang="EN-US">&nbsp;</span></p>
+<p style='text-indent:36.0pt'><span lang=EN-US>&nbsp;</span></p>
 </html>