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Immunology T cell Tools

Study the balance of Tregs and Th17 cells

Expression vectors and reporters

  • Overexpress Human/Mouse Foxp3
  • Overexpress Human/Mouse RORγt
  • Foxp3, RORγt and IL-17 reporters
  • Jurkat T cell lines

Tools to study regulatory T cells (Tregs)

   

Tregs are critical for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. Tregs can be characterized by uniquely expressing the forkhead 3 transcription factor Foxp3. SBI has innovated tools to investigate the dynamics of Foxp3 in Treg cells.

Overexpress Foxp3 with lentivectors and piggyBac transposons

Use lentivectors or piggyBac transposons with dual GFP and Puro markers to stably overexpress human or mouse Foxp3. The human and mouse Foxp3 lentivectors TCL200A-1 (human) and TCL100A-1 (mouse) and the piggyBac transposon constructs TCL200PB-1 (human) and TCL100PB-1 (mouse) were transiently transfected into 293FT cells. After 24 hours, cellular lysates were prepared and Western blot analyses performed to detect the Foxp3 protein expression using an anti-Foxp3 antibody that detects both human and mouse proteins. Robust overexpression of both human (lanes 1,3) and mouse (lanes 2,4) Foxp3 proteins were observed when compared to the nontransfected cell lysate control (lane 5). Tubulin levels were determined using an anti-tubulin antibody to serve as loading controls. The lentivector and piggyBac plasmid maps and Western blot data for the Foxp3 lentivector and piggyBac transfection experiments are shown below.

Inducible overexpression of Foxp3 with the cumate switch lentivector

The all-in-one cumate switch lentivector can be used to establish stable cell lines that can be induced to overexpress Foxp3 using cumate. The mouse Foxp3 was cloned upstream of the IRES GFP cassette and was placed under the control of the upstream cumate switch promoter. The EF1 alpha promoter drives the expression of the CymR repressor-T2A-Puro cassette. The TCL500A-1 CuO-Mouse Foxp3-IRES-GFP-EF1-CymR-T2A-Puro lentivector plasmid was packaged into lentivirus and transduced into 293FT cells. A stable cell line was established using puromycin (2.5ug/ml) selection for three days. To induce the over expression of mFoxp3, 300ug/ml cumate solution was added to the media and cells imaged after 7 days. The GFP marker serves as a positive response marker to cumate induction. Cellular proteins were harvested after seven days and western blot analysis performed to test for mFoxp3 protein induction using an anti-Foxp3 antibody and also probed with an anti- tubulin antibody for protein loading controls. The GFP co-induction marker cell images and the Western blot data are depicted below.

Jurkat stable T cell lines overexpressing either human or mouse Foxp3

Human Jurkat T cells were transduced with either TCL200A-1 (human) and TCL100A-1 (mouse) packaged lentivirus and stable cell lines established using puromycin (2.5ug/ml) selection for 7 days. The resulting stable cell lines were evaluated for GFP levels using flow cytometry and cellular lysates tested using Western blots for the overexpression of Foxp3. The flow cytometry and Western blot data are shown below.