Team:SYSU-China/Project/Results

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<DIV class="chapter">
<DIV class="chapter">
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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>
<p>
<p>
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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 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 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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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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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. 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.
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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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</p>
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<div class="results_guide">
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<p>
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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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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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<a class="guide_button" id="suicidegene" href="https://2013.igem.org/Team:SYSU-China/Project/Result/element_test?#button01"></a>
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</p>
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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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<h1>The Cruel Guard：Suicide Gene and Its Ancillary Facility</h1>
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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>1.Comparison between Suicide Genes: who is the most tough killer?</h2>
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</div>
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<p>
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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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</p>
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Figure1. Apoptosis rate comparison of different Suicide Genes using DAPI staining
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<img />
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<p>
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DAPI staining was carried out for each Suicide Gene and we found that apoptosis phenotype was most outstanding for RIP1, the >>>>>>. In order to quantitate the death rate, Flow Cytometry(FCM) was performed using PI staining. The results is amazing: the >>>>>>>
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</p>
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<h2>2.Rescue scheme: a combination of Suicide Gene and MicroRNA-Target system.</h2>
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<p>
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Tough as the Suicide Gene is, what we expect from it was to protect instead of killing the innocent. This should work through cooperation of both Suicide Gene and MicroRNA-Target system: a killer and a detector. We carried out a rescue experiment with gradient-dosage MiR122 to verify that the MicroRNA-Target system do work in a proper range of MiR122 concentration. Here to work properly means it doesn’t knockdown Suicide Gene under concentration as low as cancer cell or iPSC, but can robustly repress it under concentration of liver cell. And surely we also refer to many papers of the miR122 level in different cell lines, and perform RT-qPCR to verify it.
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</p>
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Figure 2
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<h2>3.Controlling the switch: a combination of Suicide Gene and Tet-off system.</h2>
 
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Figure 3
 
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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>
 
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Because of the limitation of transient transfection method(efficiency, duration time), we don’t expect good results from this experiment. However, since establishment of stable cell line takes much time and troubleshooting, we may still do it as a complement.
 
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</p>
 
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Figure 4
 
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<h2>5.Survival experiment of liver cell.</h2>
 
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<p>
 
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With our collinear-regression model of MicroRNA-Target system, the knockdown efficiency predicted with a two-copy complete target-site and the miR122 concentration of liver cell would be >>>%, and the experiment shows that this gives a good rescue effect.
 
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</p>
 
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Figure 5
 
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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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<p>
 
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An orthogonal experiment was performed to make a comparison between the knock-down efficiency of different target-site under different concentration of MiR122. Two plasmids were co-transfected using PEI into Bosc cell line. Plasmids A expressed GFP whose mRNA carries different target-site of miR122 in its 3’ UTR. Plasmids B express MiR122. Target-site with most notable knock-down efficiency will be chosen for further characterization and construction.
 
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</p>
 
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<p>
 
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The miR122 expression plasmid used in this experiment also express a GFP, which is a bug originally neglected in our design. However, we find out that although this does interfere our results to some degree, we can still gain insight into different performance between target-sites. Also, this accident may be kind of an unexpected clue to a new discovery, which may be one of our future work>>>>
 
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</p>
 
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<p>
 
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After finding out this bug, we do quick-change to introduce a stop-codon into the CDS of GFP, which proves very efficient.
 
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</p>
 
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Figure1. Comparison of performance of different target-sites in knocking down GFP expression
 
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Plasmids construction
 
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Fluorescence
 
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<img />
 
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<p>
 
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The result shows that with the same copy number, complete target-site works better than CULT target-site, which fits the results of the 2010 Heiderburg iGEM team. What’s more, two copies target-site works better than single site, which encourage us to further build target-sites of more copy number and quantitatively test their performance.
 
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</p>
 
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<h2>2.Quantitative characterization of the complete-target-site series and the linear-regression model.</h2>
 
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<p>
 
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In order to build a precisely controlled device, we decided to further characterized the complete-target-sites of 1\2\4 copy number and chose the most suitable one for pathway construction. To begin with we design another orthogonal experiment for the two variables, target-site-number N and MiR122 concentration M, with a more precise gradients for MiR122. But soon we found that would be too labour-costing. A better way is to replace it with 2 dosage-gradient experiment and a mathematic model.
 
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</p>
 
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<u><em>A.Dosage experiment testing performance of 2 copies complete target-site</em></u>
 
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<p>
 
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Plasmids A was the same as used in experiment 1, while plasmids B no longer expressed GFP due to the silencing mutation in CDS. In order to quantitate target-site performance under different concentration of MiR122, a dosage-gradient of plasmids B was set up and so was a parallel experiment groups to leave enough samples of each concentration for both Western Blot and RT-qPCR.
 
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</p>
 
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Figure2.
 
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Fluorescence
 
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Western Blot of GFP
 
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RT-qPCR of MiR122.
 
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<img />
 
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Knock down efficiency chart
 
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<p>收样时间：48hr<br />Target-site: Complete 2-copies</p>
 
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<table width="536">
 
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<tr>
 
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<td>Mir122</td>
 
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<td>1</td>
 
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<td>4</td>
 
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<td>10</td>
 
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<td>28</td>
 
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<td>76</td>
 
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<td>161</td>
 
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</tr>
 
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<tr>
 
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<td>GFP(Western Blot)</td>
 
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<td></td>
 
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<td></td>
 
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<td></td>
 
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<td></td>
 
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<td></td>
 
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<td></td>
 
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</tr>
 
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</table>
 
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<p>
 
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The results show that MiR122 does have an dosage effect upon target-site. The concentration that have the best knock-down performance is about >>>>>, and RT-qPCR of different kinds of cell line shows that only liver cell is able to turn off this second switch of iPS-Safeguard and avoid the fate of apoptosis.
 
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</p>
 
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<p>
 
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<u><em>B.Performance of complete target-site of different copy number</em></u>
 
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</p>
 
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<p>
 
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To further characterized the several candidate target-site for our iPS-Safeguard, we design another gradient experiment and this time let target-site copy number be the variable. The MiR122 concentration is fixed at one that gives a best performance and since we’ve already known the relationship between plasmids concentration and MiR122 expression level, only a Western-Blot is needed.
 
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</p>
 
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Figure3.
 
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Fluorescence
 
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Western blot
 
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Knock down efficiency chart
 
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<p>Mir122 concentration:0.75ug=</p>
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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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<table width="536">
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<tr>
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<td>Target Site Number</td>
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<td>0</td>
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<td>1</td>
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<td>2</td>
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<td>4</td>
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</tr>
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<tr class="first-table-line">
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<td>GFP(Western Blot)</td>
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<td></td>
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<td></td>
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<td></td>
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<td></td>
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</tr>
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</table>
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<p>
<p>
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The results show that 4 copy target-site has the most brilliant knock-down efficiency, which is about〉〉〉〉, which conforms to the data given by 2010 Heiderburg using dual-fluorescent system. However, since we are on a tight schedule in this competition, we can’t wait until this results and have already adopt the 2 copy target-site for further construction. Has we needed a better knock-down efficiency later, we can replace the target-site conveniently with our restriction enzyme.
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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.  
</p>
</p>
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<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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C. the linear regression model for miR122-target system.
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<div class="results_guide2">
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</p>
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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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<p>
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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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The results of experiment A and B then provide enough data for Model construction. The （拟合了线性关系，用matlab 做出了这样的一幅图？总之就是一个建模过程的简单描述）. The model shows that(关于模型说明的问题的一个精简解释，说明了什么趋势，预测了什么事情)
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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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Figure4. Linear Regression Model for MicroRNA-Target Gate.
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</div>
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</p>
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<img />
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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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<p>
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<EM>
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<STRONG>Experiment Design:</STRONG><br />
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Two plasmids were co-transfected into Bosc cell line using PEI, plasmids A expressed tTA protein and one carries the TRE-tight element and while response to tTA protein, expressed eGFP (BBa>..). The well with only plasmids B characterized the basal leaky expression of TRE-tight element, which can be viewed as the first off-state. Well with both plasmids represents the on-state of the system, and the third well with dox represents the second off-state. Dox concentration was chosen according to protocols in order to give a best performance of the system and avoid hazard on cell growth.
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</EM>
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</p>
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Figure1. Comparison of on/off switch performance between two Tet-off systems.
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Plasmids construction
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Fluorescence of on/off switch of two Tet-off systems.
 
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Western Blot of GFP expression level of on/off state of Tet-off systems.
 
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<img />
 
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<img />
 
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<p>
 
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Conclusion:<br />
 
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The data shows that the two systems have different on/off expression levels, but both shows good response to Dox, which is low off-expression-not so high on-expression, and relatively high off-expression-robust on-expression. The variability in performance can be properly utilized according to the apoptosis condition conducted by Suicide Gene.
 
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</p>
 
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<DIV id="references">
 
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<h2References</h2>
 
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<p><a class="references">[1]</a>Hui Xu etc. Liver-Enriched Transcription Factors Regulate MicroRNA-122 That Targets CUTL1 During Liver Development.</p>
 
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<p><a class="references">[2]</a>Tet-Off and Tet-On Gene Expression Systems User Manual</p>
 
</DIV>
</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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