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<h3>Part 1 :SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) </h3> | <h3>Part 1 :SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) </h3> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/4/46/T--CPU_CHINA--parts_11_1_figure_1.png |
" width = "700"></center> | " width = "700"></center> | ||
<h4 align=middle>Figure 1. The structure of SynNotch fusion protein.</h4> | <h4 align=middle>Figure 1. The structure of SynNotch fusion protein.</h4> | ||
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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/ | + | <center><img src="https://static.igem.org/mediawiki/2017/2/2b/T--CPU_CHINA--parts_11_1_figure_2.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/ | + | <center><img src="https://static.igem.org/mediawiki/2017/5/58/T--CPU_CHINA--parts_11_1_figure_3.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> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/b/b4/T--CPU_CHINA--parts_11_1_figure_4.png" width"700"></center> |
<h4 align=middle>Figure 4. The expression of Flag-FOXP3 by activating the expression of the USP7 gene in SynNotch system in inflammatory condition</h4> | <h4 align=middle>Figure 4. The expression of Flag-FOXP3 by activating the expression of the USP7 gene in SynNotch system in inflammatory condition</h4> | ||
</div> | </div> | ||
<h3>Part 2:UUpromU (<a href="http://parts.igem.org/Part:BBa_K2506004">BBa_K2506004</a>)</h3> | <h3>Part 2:UUpromU (<a href="http://parts.igem.org/Part:BBa_K2506004">BBa_K2506004</a>)</h3> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/1/13/T--CPU_CHINA--parts_11_1_figure_5.png" width = "700"></center> |
<h4 align=middle>Figure 5. The design of UUpromU and its application.</h4> | <h4 align=middle>Figure 5. The design of UUpromU and its application.</h4> | ||
</div> | </div> | ||
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<h4><br>UUpromU can synergize with SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>), activate the USP7 gene by the action of the inflammatory cytokine IL-17A and stabilize the expression of FOXP3. To verify that, we transfected three-plasmid expression system into Flag-FOXP3-Jurkat cells by lentivirus transfection and electroporation. In the presence of inflammatory cytokine IL-6, we administered IL-17A with different concentrations and detected the expression levels of USP7 and FOXP3 by immunoblotting (Figure 6). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A. It means that UUpromU can synergize with SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) and activate USP7 gene under the action of inflammatory cytokine IL-17A, thus stabilizing the effect of FOXP3.</h4> | <h4><br>UUpromU can synergize with SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>), activate the USP7 gene by the action of the inflammatory cytokine IL-17A and stabilize the expression of FOXP3. To verify that, we transfected three-plasmid expression system into Flag-FOXP3-Jurkat cells by lentivirus transfection and electroporation. In the presence of inflammatory cytokine IL-6, we administered IL-17A with different concentrations and detected the expression levels of USP7 and FOXP3 by immunoblotting (Figure 6). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A. It means that UUpromU can synergize with SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) and activate USP7 gene under the action of inflammatory cytokine IL-17A, thus stabilizing the effect of FOXP3.</h4> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/9/ | + | <center><img src="https://static.igem.org/mediawiki/2017/9/9a/T--CPU_CHINA--parts_11_1_figure_6.png" width = "700"></center> |
<h4 align=middle>Figure 6. The expression of USP 7 under the synergy of UUpromU and SynNotch system in inflammatory conditions.</h4> | <h4 align=middle>Figure 6. The expression of USP 7 under the synergy of UUpromU and SynNotch system in inflammatory conditions.</h4> | ||
</div> | </div> | ||
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<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/2/24/T--CPU_CHINA--parts_11_1_figure_7.png" width = "700"></center> |
<h4 align=middle>Figure 7. The structure of CAR fusion protein.</h4> | <h4 align=middle>Figure 7. The structure of CAR fusion protein.</h4> | ||
</div> | </div> | ||
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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/ | + | <center><img src="https://static.igem.org/mediawiki/2017/d/d2/T--CPU_CHINA--parts_11_1_figure_8.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/ | + | <center><img src="https://static.igem.org/mediawiki/2017/c/c5/T--CPU_CHINA--parts_11_1_figure_9.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> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/4/4e/T--CPU_CHINA--parts_11_1_figure_10.png" width="700"></center> |
<h4 align=middle>Figure 10. The expression of fluorescence in CAR system observed by fluorescence microscope<br> | <h4 align=middle>Figure 10. The expression of fluorescence in CAR system observed by fluorescence microscope<br> | ||
<br>For more experimental data details, please refer to our <a href="https://2017.igem.org/Team:CPU_CHINA/Results">RESULTS</a> section.</h4> | <br>For more experimental data details, please refer to our <a href="https://2017.igem.org/Team:CPU_CHINA/Results">RESULTS</a> section.</h4> | ||
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<h3>Reference:</h3> | <h3>Reference:</h3> | ||
− | + | <h4>1.Roybal KT, Williams JZ, et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 2016. doi: 10.1016/j.cell.2016.09.011.</h4><h4> | |
− | + | 2.Klebanoff CA, Restifo NP. Customizing Functionality and Payload Delivery for Receptor-Engineered T Cells. Cell 2016. doi: 10.1016/j.cell.2016.09.033.</h4><h4> | |
− | + | 3.Ellebrecht CT, Bhoj VG, et al. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease. Science 2016. doi: 10.1126/science.aaf6756.</h4><h4> | |
− | + | ||
− | + | 4.Fransson M, Piras E, et al. CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery. J Neuroinflammation. 2012. doi: 10.1186/1742-2094-9-112.</h4><h4> | |
− | + | ||
− | + | 5.MacDonald KG, Hoeppli RE, et al. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor. J Clin Invest. 2016. doi: 10.1172/JCI82771. Epub 2016 Mar 21.</h4><h4> | |
− | + | 6.Roybal KT, Rupp LJ. et al. Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits. Cell. 2016. doi: 10.1016/j.cell.2016.01.011.</h4><h4> | |
− | + | 7.Kononenko AV, Lee NC. et al. Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette. Nucleic Acids Res. 2015. doi: 10.1093/nar/gkv124.</h4><h4> | |
− | + | 8.Müller K, Zurbriggen MD, Weber W. An optogenetic upgrade for the Tet-OFF system. Biotechnol Bioeng. 2015. doi: 10.1002/bit.25562. </h4><h4> | |
− | + | 9.Khalil DN, Smith EL, et al. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol. 2016 May. doi: 10.1038/nrclinonc.2016.25. </h4><h4> | |
− | + | 10.Chen Z, Barbi J, et al. The ubiquitin ligase Stub1 negatively modulates regulatory T cell suppressive activity by promoting degradation of the transcription factor Foxp3. Immunity. 2013. doi: 10.1016/j.immuni.2013.08.006.</h4><h4> | |
− | + | 11.Van Loosdregt J, Fleskens V, et al. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity. 2013. doi: 10.1016/j.immuni.2013.05.018.</h4><h4> | |
− | + | 12.Wang L, Kumar S, et al. Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3+ T-regulatory Cell Function and Promotes Antitumor Immunity. EBioMedicine. 2016. doi: 10.1016/j.ebiom.2016.10.018.</h4><h4> | |
− | + | 13.Chen X, Oppenheim JJ. Th17 cells and Tregs: unlikely allies. J Leukoc Biol. 2014 May;95(5):723-731. Epub 2014 Feb 21</h4><h4> | |
− | + | 14. Ren J, Li B. The Functional Stability of FOXP3 and RORγt in Treg and Th17 and Their Therapeutic Applications. Adv Protein Chem Struct Biol.2017;107:155-189. doi: 10.1016/ bs.apcsb.2016.10.002. Epub 2016 Dec 15.</h4><h4> | |
+ | 15. Chen X, Oppenheim JJ. Th17 cells and Tregs: unlikely allies. J Leukoc Biol. 2014 May;95(5):723-731. Epub 2014 Feb 21.</h4><h4> | ||
+ | 16.Brudno JN, Kochenderfer JN. Chimeric antigen receptor T-cell therapies for lymphoma. Nat Rev Clin Oncol. 2017 Aug 31. doi: 10.1038/nrclinonc.2017.128.</h4><h4> | ||
+ | 17.Miossec P, Kolls JK. Targeting IL-17 and TH17 cells in chronic inflammation. Nat Rev Drug Discov. 2012 Oct;11(10):763-76. doi: 10.1038/nrd3794.</h4><h4> | ||
+ | 18.Mills KH. TLR-dependent T cell activation in autoimmunity. Nat Rev Immunol. 2011 Nov 18;11(12):807-22. doi: 10.1038/nri3095.</h4><h4> | ||
+ | 19.Burzyn D, Benoist C, Mathis D. Regulatory T cells in nonlymphoid tissues. Nat Immunol. 2013 Oct;14(10):1007-13. doi: 10.1038/ni.2683. Epub 2013 Sep 18.</h4><h4> | ||
+ | 20.Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008 May 30;133(5):775-87. doi: 10.1016/j.cell.2008.05.009.</h4> | ||
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 15:12, 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 |