Team:SYSU-China/Project/Results

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<span>UPDATE <INS>09/22/2013</INS></span>
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<span>Project/Results</span>
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<h1>An Introduction to Experiments Designing</h1>
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<h1>Overview of Results</h1>
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<p>Our experimental results are presented by two lines. One is the <strong>test for each part</strong>, the other is the <strong>test in each period of cells</strong>.    </p>
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<h2><a href="https://2013.igem.org/Team:SYSU-China/Project/Result/element_test?#button02">Test for each part </a></h2>
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The design of iPSC Safeguard pathway is simple and elegant. Basically, we can divide them into three major devices—the Suicide Gene, the MicroRNA-Target system and the Tet-off system, which
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Our iPSC safeguard device consists of three parts: the killer (suicide gene), the sensor (miR-122 binding site) and the switch (Tet-off system). Candidates of each part 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>
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<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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<div class="results_guide">
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<a class="guide_button" id="target" href="https://2013.igem.org/Team:SYSU-China/Project/Result/element_test?#button02"></a>
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<a class="guide_button" id="suicidegene" href="https://2013.igem.org/Team:SYSU-China/Project/Result/element_test?#button01"></a>
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<a class="guide_button" id="tet-off" href="https://2013.igem.org/Team:SYSU-China/Project/Result/element_test?#button03"></a>
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<a class="guide_button" id="lenti-virus" href="https://2013.igem.org/Team:SYSU-China/Project/Result/element_test?#button04"></a>
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has been described in detail in the Design module. For each device we had several candidates and before we finally assemble them into the whole pathway, we decided to test and characterize
 
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them carefully in the first place. The accurately quantitated parameters of each device then helped us set up a model and predicted which assembly scheme would work out best.
 
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In order to test the devices, elements were cloned into our two major plasmids backbone: pcDNA3.0(from Invitrogen) and p199(from。。。).The maps or design are shown in each parts of Results.
 
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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,
 
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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
 
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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
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<h2><a href="https://2013.igem.org/Team:SYSU-China/Project/result/stable_assay?#button01">Test in different cells</a> </h2>
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with miR122 target-site.
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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>
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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
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<div class="results_guide2">
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<a class="guide_button2" id="ips" href="https://2013.igem.org/Team:SYSU-China/Project/result/stable_assay?#button01"></a>
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<a class="guide_button2" id="hepatocyte" href="https://2013.igem.org/Team:SYSU-China/Project/result/stable_assay?#button02"></a>
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<a class="guide_button2" id="hepatoma" href="https://2013.igem.org/Team:SYSU-China/Project/result/stable_assay?#button03"></a>
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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
 
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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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Latest revision as of 03:36, 29 October 2013

ipsc

Project/Results

Overview of Results

Our experimental results are presented by two lines. One is the test for each part, the other is the test in each period of cells.

Test for each part

Our iPSC safeguard device consists of three parts: the killer (suicide gene), the sensor (miR-122 binding site) and the switch (Tet-off system). Candidates of each part 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.

Test in different cells

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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