BaoqiangCPU (Talk | contribs) |
|||
Line 26: | Line 26: | ||
<h4><br>To test the feasibility of transfecting multiple plasmids into Treg cells, we acquired the Flag-FOXP3-Jurkat cell line from Shanghai Institute of Immunology, Medical College, Shanghai Jiao Tong University. As a stable transfection strain with high expression of Flag-FOXP3 obtained by transfecting Flag-FOXP3 fusion protein into Jurkat T cells, it is a decent model to simulate the Treg status in human body. In our experiment, we introduced our three-plasmid expressing system into the Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of both SynNotch and CAR system in Flag-FOXP3-Jurkat cells were confirmed by western blot and quantitative real-time PCR (Figure 1).</h4> | <h4><br>To test the feasibility of transfecting multiple plasmids into Treg cells, we acquired the Flag-FOXP3-Jurkat cell line from Shanghai Institute of Immunology, Medical College, Shanghai Jiao Tong University. As a stable transfection strain with high expression of Flag-FOXP3 obtained by transfecting Flag-FOXP3 fusion protein into Jurkat T cells, it is a decent model to simulate the Treg status in human body. In our experiment, we introduced our three-plasmid expressing system into the Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of both SynNotch and CAR system in Flag-FOXP3-Jurkat cells were confirmed by western blot and quantitative real-time PCR (Figure 1).</h4> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> |
<h4 align=middle>Figure1. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation</h4> | <h4 align=middle>Figure1. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation</h4> | ||
</div> | </div> | ||
Line 34: | Line 34: | ||
<h4><br>To explore the effect of IL-17A concentration on the function of SynNotch with the presence of IL-6, various concentrations of IL-17A were given and the expression of USP7 and FOXP3 was tested. With a higher concentration of IL-17A, a higher expression of USP7 and FOXP3 was detected, indicating that the concentration of IL-17A played an important role in the expression level of SynNotch.</h4> | <h4><br>To explore the effect of IL-17A concentration on the function of SynNotch with the presence of IL-6, various concentrations of IL-17A were given and the expression of USP7 and FOXP3 was tested. With a higher concentration of IL-17A, a higher expression of USP7 and FOXP3 was detected, indicating that the concentration of IL-17A played an important role in the expression level of SynNotch.</h4> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/3/3a/T--CPU_CHINA--results_fig2.png" width = "800"></center> |
<h4 align=middle>Figure2. The SynNotch system stabilizing FOXP3 in Treg under inflammatory conditions | <h4 align=middle>Figure2. The SynNotch system stabilizing FOXP3 in Treg under inflammatory conditions | ||
Revision as of 14:59, 31 October 2017