Nuclear Receptor Analysis Luciferase Vectors

Vectors To Bind Nuclear Receptors Using Recognition Sequences or One-Hybrid Assay

• Create a binding domain fusion with the pFN26 (BIND) hRluc-neo Flexi® Vector and use with the pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector
• Mouse mammary tumor virus long terminal repeat in pGL4.36[luc2P/MMTV/Hygro] Vector assesses androgen or glucocorticoid responses
• The estrogen receptor ligand binding domain (pBIND-Er∝) and the glucocorticoid receptor ligand binding domain (pBIND-GR) are already cloned

Nuclear receptor analysis can be performed with traditional means using a minimal promoter vector with nuclear receptor response elements upstream. Alternatively, you can use viral elements like the mouse mammary tumor virus long terminal repeat promoter in the pGL4.36[luc2P/MMTV/Hygro] Vector to judge androgen or glucocorticoid responses. In many cases, using these methods requires a cell line with the appropriate endogenous nuclear receptors, meaning you may need different cell lines for each nuclear receptor study. However, a method using the principles of the yeast two-hybrid system was adapted for nuclear receptor work. The nuclear receptor ligand binding domain is fused to the GAL4 DNA binding domain in the pFN26A (BIND) hRluc-neo Flexi® Vector and co-transfected with pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector, a firefly luciferase reporter vector containing repeats of the GAL4 upstream activation sequence upstream of a minimal promoter. The ligand binding domain is responsible for ligand binding, homo- or heterodimerization and interactions with co-activator or co-repressors. The one-hybrid method allows you work with any cell line and nuclear receptor you desire. Two pFN26A vectors containing the estrogen receptor ligand binding domain (pBIND-ERα) and the glucocorticoid receptor ligand binding domain (pBIND-GR) are available for use in the one-hybrid method.

A GAL4-based system removes background signals from endogenous receptors, and the optimized 9X GAL4 UAS improves cell responses with better signal:noise ratio. The combination Renilla/Neomycin marker offers normalization with Dual-Luciferase® Assay or use of a selectable marker for generating stable cell lines, all with one vector. This one-hybrid assay means you can compare or profile all nuclear receptors with a single experimental system. The destabilized and optimized luc2P luciferase gene offers greater sensitivity and shorter induction times in the reporter assay.

Applications

• Discover nuclear receptor ligands and modulators.
• Drug discovery/HTS.
• Cancer research.
• Inflammation research.
• Environmental toxicology.

Reference

1. Evans, R.M. (1988) The steroid and thyroid hormone receptor superfamily. Science 240, 889–95.

ItemPart #Size

pFN26A (BIND) hRluc-neo Flexi® Vector

E138A1 × 20μg

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