Team:SYSU-China/Project/Results

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<DIV class="chapter">
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<span>UPDATE <INS>09/23/2013</INS></span>
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<span>Project/Results</span>
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<h1>1 compatability test in hepatocytes(in progress)</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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One of the aims of our project is to build a pathway that can be sustained in iPSC-derived hepatocytes and remains functional to eliminate spontaneous cancerous cells. As proven previously, miR-122 targeted suicide pathway will be knock down in cells with high level of miR-122 (in our test, miR-122 level as high as… is adequate to knockdown …percent of gene expression which is far more than enough to function in hepatocytes with endogenous miR-122). We still need to confirm this result in hepatocytes. </p>
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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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<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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Mouse primary hepatocytes are successfully separated and then infected with our pathway by lentivirus. Since primary hepatocytes is difficult to be transfected, we have used lentivirus under ultracentrifugation to increase the efficiency of integration. The design of our pathway in lentivirus is described in …page. Generally, it takes 2 weeks to complete selecting for a constant expression cell line. Because our tet-off system resides in 2 independent plasmids, it takes additional time to finish the successive antibiotic selection for both 2 modules.
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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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We still need to test whether this pathway will interfere the normal function of hepatocytes by doing…
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<h1>The Cruel Guard：Suicide Gene and Its Ancillary Facility</h1>
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<h2>1.Comparison between Suicide Genes: who is the most tough killer?</h2>
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We collected several different Suicide Genes which functions in different pathways and patterns. In order to choose one that is most capable of inducing apoptosis in cancer cell, we first carried out a comparison experiment for these genes.
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<h1>2 pluripotent test in iPSCs( in progress)</h2>
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Our pathway is designed to be inplanted in the iPSC-induction phase and begin to function after redifferentiation. It should reach the following standards:
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<li> promoter is functional during the time of redifferentian</li>
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<li>  leakage expression is tolerable</li>
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<li> pluripotent remains in iPSCs with pathway</li>
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<li>directional redifferention should be increased</li>
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<li> our pathway can kill transformed cancerous cell</li>
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<li> our pathway is functional in long term</li>
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<h1>3 fine-tuning of parts</h1>
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<h1>4 directional differention test in iPSC</h1>
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<h1>5 tumorgensis test in iPSCs</h1>
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<h1>Discussion</h1>
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<p>
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Include several standard questions and answers frequently asked.
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</p>
</p>
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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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<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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</DIV>
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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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