Description:

Principle:

NFkB plays an important role in controlling many biological processes including immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. In response to the various stimuli, such as stress, cytokines, free radicals, ultraviolet irradiation, and bacterial or viral antigens, NFkB is activated and translocate from cytoplasm to nucleus, where NFkB binds to its response element on the promoter region and regulates a wide spectrum of gene expression. Dysfunction of NFkB activity is associated with cancer, inflammatory and autoimmune disease, and viral infection. Monitoring the NFkB activity is essential to unveil the mechanism of these diseases and conduct drug discovery. Signosis has established an NFkB luciferase reporter stable cell line that has been stably transfected with pTA-NFkB-luciferase reporter vector, which contains 4 repeats of NFkB binding sites, a minimal promoter upstream of the firefly luciferase coding region.Therefore, the cell line can be used as a reporter system for monitoring the activation of NFkB triggered by stimuli treatment, enforced gene expression and gene knockdown.The stable cell line can be used for studying NFkB signaling pathways activated by different cytokines, such as TNFa,and many other stimuli.

The cell line was established by transfection using apTA-NFkB-luciferase reporter vector, which contains 4 repeats of NFkB binding sites, a minimal promoter upstream of the firefly luciferase coding region,along with hygromycin expression vector followed by hygromycin selection. The hygromycin resistant clones were subsequently screened for TNFa-induced (or IL-1) luciferase activity.

Principle behind TF luciferase reporter. TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression. The promoter region can consists of multiple repeats of a cis-element TF binding site, a DNA fragment from the promoter region of a known TF downstream gene, or a DNA fragment containing putative/known TF binding sites. There are several ways that a TF can be activated, such as through extracellular stimuli or through intracellular signaling pathways. Once activated, the TF translocates to the nucleus and often interacts with relevant co-factors to drive gene expression. Once luciferase is expressed, it can generate light in an enzymatic assay and the amount of light measured is positively correlated with the level of TF activation.

Data:

Analysis of TNFa/NFkB Pathway Reporter Cell Line in response to TNFa treatment.The cells were seeded on a 96-well plate for overnight with DMEM including 10% FBS. The cell then were treated with 20ng/ml TNFa in DMEM + 0.1% FBS for 6 hours.