Description(s):

NFAT Luciferase Reporter HEK293 Stable Cell Line is derived from human embryonic kidney, and stably express firefly luciferase reporter gene under the control of NFAT response element.  This cell line is an ideal cellular model for monitoring the activation  of Calcium Signaling Receptor Signaling Pathway triggered by stimuli treatment, enforced gene expression, and gene knockdown.

Principle

NFATs are a family of transcriptional factors that play an important role in immune response as well as in the development of cardiac, skeletal muscle, and nervous systems.  NFATs are regulated by calcium signaling. Through calcium activation of the phosphatase calcineurin, NFATc proteins translocate from the cytoplasm into the nucleus, where they cooperate with other proteins to mediate gene expression. Nuclear import of NFAT is blocked by kinases such as PKA and GSK3. NFATs are also implicated in breast cancer.  Signosis has established NFAT luciferase reporter HEK293 cell line that has been stably transfected with a NFAT-luciferase reporter construct. Via the analysis of luciferase, the cell line can be employed to monitor the cellular changes of NFAT activities that are triggered by stimulation, compound treatment, enforced gene expression, or gene knockdown.

The cell line is established by transfection using a pTA-NFAT-luciferase reporter vector, which contains NFAT 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 PMA+ionomycin-induced luciferase activity.

 

The principle behind TF luciferase reporter.  TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression.  The promoter region can consist 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 NFAT Luciferase Reporter HEK293 Stable Cell Line.  The cells were seeded on a 96-well plate overnight with DMEM including 10% FBS. The cells then were treated with or without 10ng/ml PMA and 1uM Ionomycin in DMEM and 0.1% FBS for 18 hours. Data was read on a luminometer with a sensitivity of 3x10^-21 moles luciferase.

Literature

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