Team:SYSU-China/Project/Results

From 2013.igem.org

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In order to test the devices, elements were cloned into our two major plasmids backbone: pcDNA3.0 and p199.The maps or design are shown in each parts of Results. We drove the Suicide Gene with CMV promoter to ensure a robust expression and prominent phenotype in cells. An eGFP was used to indicate the performance of Tet-off and MicroRNA-Target system, which could be quantitated through Image J and Western-Blot. All separately testing experiments were carried out via transient transfection in Bosc and HepG2, considering both the transfection efficiency and representation of liver cancer cell. Also, a survival experiment was done on liver cell to ensure that its miR122 level can successfully knockdown Suicide Gene with miR122 target-site.</p>
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Our iPSC safeguard consists of three devices: the suicide gene, the miR-122 target and the Tet-off system. Candidates of each device were cloned and characterized. For Suicide Gene, RIP1 and RIP3 has optimal lethal effect in both HEK293 and HepG2 while apoptin can only kill HepG2. MiR-122 target can sensitively regulate gene expression as GFP level negatively correlates with exogenous level of miR-122 transected to HEK293. Tet-off system with pTight and tTA shows a low leakage expression level and medium full expression level. Differential equations were employed to analyze and predict the performance of the pathway.</p>
<p><strong>If you want to see the results for testing each parts, please click the following wheer gears.</strong></p>
<p><strong>If you want to see the results for testing each parts, please click the following wheer gears.</strong></p>
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After confirmation of each device, we proceeded to working on iPSC and assembly of the whole pathway. The whole pathway was integrated into two p199 plasmids, one for regulating and one for response .Lenti-virus packaging these two plasmids was produced and 3 cell lines of iPSC, HepG2, Hela were transfected. This successfully gave us the stable cell line with our iPSC Safeguard design. Then further test and characterization of the pathway's performance of working as a whole were carried out. All the data can be seen in >>>>>.
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After prove of function, the pathway was packaged into lentivirus to test the performance in our three destiny cell types: iPSCs, hepatocytes and hepatomas. So far, we have successfully generated Oct4-GFP miPSCs with pluripotency verified. All three suicide genes have significant lethal effects in iPSCs and HepG2. With pathway integrated into the genome by lentivirus, leakage expression of TRE3G is low enough to be safe, but the pTight does not work well. Doxycycline has no obvious side effects to iPSC. Stable cell line with both TRE3G and tTA requires  a little more time to finish the second round of selection and we are still waiting for result of teratoma formation from our stable iPSCs.  
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<p><strong>If you want to see the results testing iPSCs in different periods, please click the following cute cells.</strong></p>
<p><strong>If you want to see the results testing iPSCs in different periods, please click the following cute cells.</strong></p>

Revision as of 18:37, 27 September 2013

ipsc

UPDATE 09/22/2013

Overview of result

Our iPSC safeguard consists of three devices: the suicide gene, the miR-122 target and the Tet-off system. Candidates of each device were cloned and characterized. For Suicide Gene, RIP1 and RIP3 has optimal lethal effect in both HEK293 and HepG2 while apoptin can only kill HepG2. MiR-122 target can sensitively regulate gene expression as GFP level negatively correlates with exogenous level of miR-122 transected to HEK293. Tet-off system with pTight and tTA shows a low leakage expression level and medium full expression level. Differential equations were employed to analyze and predict the performance of the pathway.

If you want to see the results for testing each parts, please click the following wheer gears.

After prove of function, the pathway was packaged into lentivirus to test the performance in our three destiny cell types: iPSCs, hepatocytes and hepatomas. So far, we have successfully generated Oct4-GFP miPSCs with pluripotency verified. All three suicide genes have significant lethal effects in iPSCs and HepG2. With pathway integrated into the genome by lentivirus, leakage expression of TRE3G is low enough to be safe, but the pTight does not work well. Doxycycline has no obvious side effects to iPSC. Stable cell line with both TRE3G and tTA requires a little more time to finish the second round of selection and we are still waiting for result of teratoma formation from our stable iPSCs.

If you want to see the results testing iPSCs in different periods, please click the following cute cells.



Sun Yat-Sen University, Guangzhou, China

Address: 135# Xingang Rd.(W.), Haizhu Guangzhou, P.R.China

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