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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<h1>The Cruel Guard</h1><h1>：Suicide Gene and Its Ancillary Facility</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>1.Comparison between Suicide Genes: who is the most tough killer?</h2>
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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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<h2>2.Rescue scheme: a combination of Suicide Gene and MicroRNA-Target system.</h2>
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<h2>3.Controlling the switch: a combination of Suicide Gene and Tet-off system.</h2>
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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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<h2>4.The final test-run: combination of all three systems before setting up stable cell line.</h2>
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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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<h2>5.Survival experiment of liver cell.</h2>
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<h1>Simulated Testing of MicroRNA-Target Devices: Security Gate for Liver Cell.</h1>
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<h2>1.Performance comparison between two kinds of target-sites: CULT and Complete<a class="quote">[1]</a>.</h2>
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<h2>2.Quantitative characterization of the complete-target-site series and the linear-regression model.</h2>
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<h1>Dosage test of Tet-off system: Controlling Panel For iPSC.</h1>
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<h2>1.Trial test of two different Tet-off system: the switching performance between on and off states.</h2>
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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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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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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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