The JAK (Janus kinase) /STAT (Signal Transducer and Activator of Transcription) pathway is activated by various cytokines and growth factors and plays a critical role in cell growth, hematopoiesis, and immune response. In mammals, there are four JAKs (JAK1, JAK2, JAK3 and TYK2) and seven STAT proteins. Interferon alpha (IFNα) is a Type I interferon. Binding of IFNα to its receptor leads to the activation of JAK1 and TYK2, which in turn phosphorylate and activate STAT1 and STAT2. The phosphorylated STAT1 and 2 form a heterodimer and bind to IRF9/p48, forming a protein complex ISGF3. This complex translocates to the nucleus and binds to the ISRE (Interferon Stimulated Response Element) in the promoter region thereby promoting transcription of interferon-inducible genes.
The ISRE Transient Pack is designed to provide the tools necessary for transiently transfecting and monitoring the activity of Type I interferon-induced JAK/STAT signaling pathway in cultured HEK293 cells. The pack contains transfection-ready vectors containing firefly luciferase as a JAK/STAT pathway-responsive reporter and constitutively expressing Renilla luciferase as a transfection control. It also includes the Dual Luciferase detection reagents to detect both luciferase activities and specialized medium for growing and assaying HEK293 cells.
The key to the ISRE Transient Pack is the ISRE luciferase reporter vector, which is a JAK/STAT pathway-responsive reporter. This reporter contains a firefly luciferase gene under the control of multimerized ISRE responsive element located upstream of a minimal promoter. The ISRE reporter is premixed with constitutively-expressing Renilla luciferase vector, which serves as an internal control for transfection efficiency.
The pack also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical to determining pathway specific effects and background luciferase activity.
Additionally, the pack includes cell culture medium (BPS Medium 1) that has been optimized for use with HEK293 cells*. BPS Medium 1 includes MEM medium, 10% fetal bovine serum, 1% non-essential amino acids, sodium pyruvate, and 1% Pen/Strep. Finally, the pack provides the Dual Luciferase (Firefly-Renilla) Assay System. These luciferase reagents provide highly sensitive, stable detection of firefly luciferase activity and Renilla luciferase activity. The dual luciferase reagents can be used directly in cells in growth medium, and can be detected with any luminometer; automated injectors are not required.
*Note: the kit may be used with other cell lines than HEK293, but an alternate cell culture medium may be required for optimal cell growth.
