Description(s):

 The GAL4-UAS system is a powerful technique for studying gene expression and function. It has also been adapted to study nuclear receptor chemical-binding functions. Our PPAR-delta LBD-driven GAL4 reporter HEK 293 stable cell line is a reliable, rapid, and sensitive method for analyzing the intracellular status of NRs upon exposure to drug candidates or other stimuli. These functional assays allow for measurement of receptor activation or inhibition by compounds and can be used to determine compound potency and selectivity.

Principle

Nuclear hormone receptors (NHRs) are a group of ligand-binding transcription factors (TFs).  More than 350 NHRs are available in the PDB. Like other TFs, they are able to regulate gene expression by binding to specific DNA regulatory elements, but their activities are modulated by the corresponding ligands. They play many important physiological functions such as embryonic development, organ physiology, cell differentiation and homeostasis. They are also associated with human diseases including cancer, obesity/diabetes, arthritis and asthma. Members of this family are often chosen to be drug targets.

Conventionally, the response element-luciferase reporter assays are used to study endogenous or exogenous receptors, and analyze the receptor signaling pathway in a native biological context.  Due to their low sensitivity of these assays and the similar response element shared by multiple NHRs, their applications are limited.  Signosis has developed LDB-driven GAL4 reporter HEK 293 stable cell lines by delivering two vectors, one expressing 8 copies of GAL4 upstream activator sequences (UAS) in front of the luciferase reporter gene, another expressing Gal4 DNA Binding Domain (DBD) and PPAR-delta ligand binding domain(LBD). Upon addition of a corresponding ligand, the DBD-LBD fusion protein is activated, and binds to GAL promoter binding site, leading to the induction of luciferase.  The stable clones were selected with hygromycin and G418, The functional assay was subsequently conducted by GW501516 treatment, and the clone with the highest induction fold has been chosen for this product. The advantages of these stable cell lines are low cross-reactivity with other nuclear receptors and less toxicity of the chimeric receptors to the cells when overexpressed.  These cell lines can be used to screen drug compounds for agonist or antagonist hit identification.

 

 

Data

Analysis of PPAR-delta Luciferase Reporter Cell Line in response to varying doses of GW501516 treatment. 
The cells were seeded on a 96-well plate overnight with DMEM including 10% FBS. The cell then were treated with varying doses of GW501516 in DMEM + 0.1% FBS for 16 hours.