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The released Gal4-VP64 will recognize UAS sequence in the upstream of promoter USP7 (in our another part <a href="http://parts.igem.org/Part:BBa_K2506004">BBa_K2506004</a>) and then these two proteins combine together, which enable USP7 gene express with high efficiency. USP7 proteins will deubiquitinate the ubiquitinated FOXP3, so that to enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. In this way, Treg cells can survive and play a role of immunosuppressor.</h4> | The released Gal4-VP64 will recognize UAS sequence in the upstream of promoter USP7 (in our another part <a href="http://parts.igem.org/Part:BBa_K2506004">BBa_K2506004</a>) and then these two proteins combine together, which enable USP7 gene express with high efficiency. USP7 proteins will deubiquitinate the ubiquitinated FOXP3, so that to enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. In this way, Treg cells can survive and play a role of immunosuppressor.</h4> | ||
<h3>Results</h3> | <h3>Results</h3> | ||
− | <h4><br>Flag-FOXP3-Jurkat cell line is a stably transfected cell line that highly expresses Flag-FOXP3. It is established by transfecting Flag-FOXP3 fusion protein gene in Jurkat T cells, and is a good model to simulate the state of human regulatory T cells. We obtained it from the molecular immunology research group of Shanghai Institute of Immunology, School of Medicine in Shanghai Jiao Tong University. In our experiment, we transfected three- plasmid expression system into Flag-FOXP3-Jurkat cells through the lentivirus-mediated gene transfer system and electroporation respectively. The expression of the SynNotch system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR ( | + | <h4><br>Flag-FOXP3-Jurkat cell line is a stably transfected cell line that highly expresses Flag-FOXP3. It is established by transfecting Flag-FOXP3 fusion protein gene in Jurkat T cells, and is a good model to simulate the state of human regulatory T cells. We obtained it from the molecular immunology research group of Shanghai Institute of Immunology, School of Medicine in Shanghai Jiao Tong University. In our experiment, we transfected three- plasmid expression system into Flag-FOXP3-Jurkat cells through the lentivirus-mediated gene transfer system and electroporation respectively. The expression of the SynNotch system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 3).</h4> |
<div> | <div> | ||
<center><img src="https://static.igem.org/mediawiki/2017/3/30/T--CPU_CHINA--part_image002.png" width = "700"></center> | <center><img src="https://static.igem.org/mediawiki/2017/3/30/T--CPU_CHINA--part_image002.png" width = "700"></center> | ||
− | <h4 align=middle>Figure 2. The | + | <h4 align=middle>Figure 2. The plasmid of the SynNotch system</h4> |
</div> | </div> | ||
<div> | <div> | ||
<center><img src="https://static.igem.org/mediawiki/2017/2/26/T--CPU_CHINA--part_image003.png" width = "700"></center> | <center><img src="https://static.igem.org/mediawiki/2017/2/26/T--CPU_CHINA--part_image003.png" width = "700"></center> | ||
− | <h4 align=middle>Figure 3. The expression of the SynNotch system in Flag-FOXP3-Jurkat cell transfected by electroporation</h4> | + | <h4 align=middle>Figure 3. The expression of the SynNotch system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection and electroporation</h4> |
</div> | </div> | ||
<h4><br>In order to investigate the relationship between the concentration of IL-17A ad the level of activity of the SynNotch system in the presence of inflammatory cytokine IL-6, we administered different concentrations of IL-17A and detected the expression levels of USP7 and FOXP3 (Figure 4). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A, which indicates that IL-17A concentration is an important factor affecting the expression level of SynNotch system. This verifies that our SynNotch system has worked well.</h4> | <h4><br>In order to investigate the relationship between the concentration of IL-17A ad the level of activity of the SynNotch system in the presence of inflammatory cytokine IL-6, we administered different concentrations of IL-17A and detected the expression levels of USP7 and FOXP3 (Figure 4). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A, which indicates that IL-17A concentration is an important factor affecting the expression level of SynNotch system. This verifies that our SynNotch system has worked well.</h4> | ||
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In addition, we linked the fluorescent protein mCherry at the C-terminus of the CAR system to facilitate the detection of gene expression. In order to accomplish this goal, we optimized the codon on <a href="http://parts.igem.org/Part:BBa_K106004">BBa_K106004</a> and connected it to the C-terminus of the CAR system.</h4> | In addition, we linked the fluorescent protein mCherry at the C-terminus of the CAR system to facilitate the detection of gene expression. In order to accomplish this goal, we optimized the codon on <a href="http://parts.igem.org/Part:BBa_K106004">BBa_K106004</a> and connected it to the C-terminus of the CAR system.</h4> | ||
<h3>Result</h3> | <h3>Result</h3> | ||
− | <h4><br>Flag-FOXP3-Jurkat cell line is a stably transfected cell line that highly expresses Flag-FOXP3. It is established by transfecting Flag-FOXP3 fusion protein gene in Jurkat T cells, and is a good model to simulate the state of human regulatory T cells. We obtained it from the molecular immunology research group of Shanghai Institute of Immunology, School of Medicine in Shanghai Jiao Tong University. In our experiment, we transfected three- plasmid expression system into Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of the CAR system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR ( | + | <h4><br>Flag-FOXP3-Jurkat cell line is a stably transfected cell line that highly expresses Flag-FOXP3. It is established by transfecting Flag-FOXP3 fusion protein gene in Jurkat T cells, and is a good model to simulate the state of human regulatory T cells. We obtained it from the molecular immunology research group of Shanghai Institute of Immunology, School of Medicine in Shanghai Jiao Tong University. In our experiment, we transfected three- plasmid expression system into Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of the CAR system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 9). We also observed a red fluorescence under a fluorescence microscope (Figure 10).</h4> |
<div> | <div> | ||
<center><img src="https://static.igem.org/mediawiki/2017/2/27/T--CPU_CHINA--part_image008.png" width="700"></center> | <center><img src="https://static.igem.org/mediawiki/2017/2/27/T--CPU_CHINA--part_image008.png" width="700"></center> | ||
− | <h4 align=middle>Figure 8. The | + | <h4 align=middle>Figure 8. The plasmid of the CAR system </h4> |
</div> | </div> | ||
<div> | <div> | ||
<center><img src="https://static.igem.org/mediawiki/2017/4/46/T--CPU_CHINA--part_image009.png" width="700"></center> | <center><img src="https://static.igem.org/mediawiki/2017/4/46/T--CPU_CHINA--part_image009.png" width="700"></center> | ||
− | <h4 align=middle>Figure 9. The expression of the CAR system in Flag-FOXP3-Jurkat cell transfected by electroporation</h4> | + | <h4 align=middle>Figure 9. The expression of the CAR system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection and electroporation</h4> |
</div> | </div> | ||
<div> | <div> |
Revision as of 11:20, 1 November 2017
PARTS
Parts Table
The parts we submitted are listed at the table below.
Part Name | Description | Design | Length |
---|---|---|---|
BBa_K2506001 | CDS of SynNotch fusion protein | Qihang Zhao | 2619bp |
BBa_K2506002 | CDS of CAR fusion protein | Qihang Zhao | 2313bp |
BBa_K2506004 | Human USP7 gene promoter and UAS sequence | Qihang Zhao | 2153bp |