BioCat GmbH • Im Neuenheimer Feld 584 • D-69120 Heidelberg • Tel.: +49(0)6221 7141516
Home -> Cell Biology -> T Cell Related Products -> Regulatory T Cell (Treg Cells) and T Cell Helper 17 (Th17 Cells) Protein Overexpression Constructs

Regulatory T Cell (Treg Cells) and T Cell Helper 17 (Th17 Cells) Protein Overexpression Constructs

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.

Image

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.

Image

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.

Image



Tools to study Th17 cells (Tregs)
T cell helper 17 (Th17) cells play a role in the inflammatory response. The characteristics of this subtype of T cell are that they secrete abundant amounts of the pro-inflammatory cytokine IL-17 and express the RORC/RORgt transcription factor.

Overexpress RORC/RORgt with lentivectors and piggyBac transposons
Use lentivectors or piggyBac transposons with dual GFP and Puro markers to stably overexpress human RORC or mouse RORãt. The mouse RORgt lentivector (TCL300A-1) and the mouse piggyBac transposon construct (TCL300PB-1) were transiently transfected into 293FT cells. After 24 hours, cellular lysates were prepared and Western blot analyses performed to detect the RORgt protein expression using a mouse RORãt antibody. Robust overexpression of mouse (lanes 1,2) RORgt proteins were observed when compared to the nontransfected cell lysate control (lane 3). 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 RORgt lentivector and piggyBac transfection experiments are shown below.

Image

Jurkat stable T cell lines overexpressing either human RORC or mouse RORgt
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 RORgt. The flow cytometry and Western blot data are shown below.

Image

 

 

Description Cat# Size Price    
CuO-Mouse Foxp3-IRES-GFP-EF1-CymR-T2A-Puro Cumate inducible lentivector plasmid TCL500A-1-SBI 10 ug 1322 € DETAILS   Add to Cart 
MSCV-Human Foxp3-EF1-GFP-T2A-Puro Stable Jurkat cell line (Puro resistant) TCL210C-1-GVO-SBI 1x10^6 cells/ml 1615 € DETAILS   Add to Cart 
MSCV-Human RORgt-EF1-GFP-T2A-Puro Stable Jurkat cell line (Puro resistant) TCL410C-1-GVO-SBI 1x10^6 cells/ml 1615 € DETAILS   Add to Cart 
MSCV-Mouse Foxp3-EF1-GFP-T2A-Puro Stable Jurkat cell line (Puro resistant) TCL110C-1-GVO-SBI 1x10^6 cells/ml 1615 € DETAILS   Add to Cart 
MSCV-Mouse RORgt-EF1-GFP-T2A-Puro Stable Jurkat cell line (Puro resistant) TCL310C-1-GVO-SBI 1x10^6 cells/ml 1615 € DETAILS   Add to Cart 
pMSCV-Human Foxp3-EF1-GFP-T2A-Puro lentivector plasmid TCL200A-1-SBI 10 ug 734 € DETAILS   Add to Cart 
pMSCV-Human Foxp3-EF1-GFP-T2A-Puro piggyBac transposon plasmid TCL200PB-1-SBI 10 ug 734 € DETAILS   Add to Cart 
pMSCV-Human RORgt-EF1-GFP-T2A-Puro lentivector plasmid TCL400A-1-SBI 10 ug 734 € DETAILS   Add to Cart 
pMSCV-Human RORgt-EF1-GFP-T2A-Puro piggyBac transposon plasmid TCL400PB-1-SBI 10 ug 734 € DETAILS   Add to Cart 
pMSCV-Mouse Foxp3-EF1-GFP-T2A-Puro lentivector plasmid TCL100A-1-SBI 10 ug 734 € DETAILS   Add to Cart 
pMSCV-Mouse Foxp3-EF1-GFP-T2A-Puro piggyBac transposon plasmid TCL100PB-1-SBI 10 ug 734 € DETAILS   Add to Cart 
pMSCV-Mouse RORgt-EF1-GFP-T2A-Puro lentivector plasmid TCL300A-1-SBI 10 ug 734 € DETAILS   Add to Cart 
pMSCV-Mouse RORgt-EF1-GFP-T2A-Puro piggyBac transposon plasmid TCL300PB-1-SBI 10 ug 734 € DETAILS   Add to Cart 

BioCat Summer Special

Summer Special

10% OFF
Norgen Sample Preparation Kits

Electrophoresis

30% OFF BioCat Universal Agarose

Email Newsletter

Subscribe to the BioCat Email Newsletter.

Logos

Logos Biosystems Cell Counters

Cell-based Assays

Cell-based assays

Choose from a wide variety of cell-based assays designed for greater accuracy and increased throughput.

50% OFF Fluorescent Proteins

Fluorescent Protein Vectors

Get 50% discount on the second and subsequent vectors when ordering two or more vectors with the same fluorescent protein.

Lentiviral Barcode Library

Lentiviral-based barcode library

Track clonal populations of cells over time or through different treatments using the lentiviral-based barcode library.

Home
Imprint / Impressum | Privacy Policy / Datenschutzerklärung
Top of Page Up!