Part:BBa_K2506001

IL-17RA_SynNotch_Gal4-VP64

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 2416
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 273
  Illegal BglII site found at 796
  Illegal BglII site found at 2202
  Illegal BamHI site found at 618
  Illegal BamHI site found at 968
  Illegal BamHI site found at 2614
  Illegal XhoI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2409
  Illegal BsaI.rc site found at 1861
  Illegal SapI site found at 1618
  

Introduction

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.

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.

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 immunosuppression.

Results

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 1 and Figure 2).

""

Figure 1 The expression of the SynNotch system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection.

Figure 2 The expression of the SynNotch system in Flag-FOXP3-Jurkat cell transfected by electroporation.

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 3). 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.

Figure 3 The expression of Flag-FOXP3 by activating the expression of the USP7 gene in SynNotch system in inflammatory condition.