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<h3>Introduction</h3> | <h3>Introduction</h3> | ||
− | <h4><br>SynNotch is a system that is capable of specifically activating the expression of the USP7 gene in the inflammatory environment, and thereby maintaining the activity of Treg cells by stabilizing the FOXP3 protein.< | + | <h4><br>SynNotch is a system that is capable of specifically activating the expression of the USP7 gene in the inflammatory environment, and thereby maintaining the activity of Treg cells by stabilizing the FOXP3 protein.</h4> |
− | In our SynNotch system, we retained the functional sequence of the transmembrane domain and the cleavage site of the Notch 1 protein. At the N-terminus, we fused the extracellular domain of IL17RA with Notch 1 to specifically recognize IL-17A secreted by Th17 cells, so that our regulatory T cells to obtain the ability to response to IL-17A. We also connected the gene of Gal4-vp64 (a fusion protein) in the downstream of Notch 1. In the presence of inflammatory cytokines IL17A, SynNotch protein is cleaved, and thus Gal4-VP64 fusion protein is detached from the cell membrane. <br> | + | <h4><br>In our SynNotch system, we retained the functional sequence of the transmembrane domain and the cleavage site of the Notch 1 protein. At the N-terminus, we fused the extracellular domain of IL17RA with Notch 1 to specifically recognize IL-17A secreted by Th17 cells, so that our regulatory T cells to obtain the ability to response to IL-17A. We also connected the gene of Gal4-vp64 (a fusion protein) in the downstream of Notch 1. In the presence of inflammatory cytokines IL17A, SynNotch protein is cleaved, and thus Gal4-VP64 fusion protein is detached from the cell membrane. </h4><h4><br> |
− | The released Gal4-VP64 will recognize UAS sequence in the upstream of promoter USP7 (in our another part BBa_K2506004 ) and then these two proteins combine together, which enable USP7 gene express with high efficiency. USP7 proteins will deubiquitinate the ubiquitinated FOXP3, so that 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 BBa_K2506004 ) and then these two proteins combine together, which enable USP7 gene express with high efficiency. USP7 proteins will deubiquitinate the ubiquitinated FOXP3, so that 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 by lentivirus transfection and electroporation respectively. The expression of the SynNotch system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 2 and Figure 3).</h4> | <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 lentivirus transfection and electroporation respectively. The expression of the SynNotch system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 2 and Figure 3).</h4> |
Revision as of 07:44, 1 November 2017
PARTS
Parts Table
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 |