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Analyzing Nuclear Receptor Signaling with Luciferase Reporters

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The pFN26A (BIND) hRluc-neo Flexi® Vector (Cat. #E1380) designed work with the pGL4.35 Vector for the one-hybrid nuclear receptor assay.

  • There are two ways to study nuclear receptor signaling:
  • cloning multiple copies of the nuclear receptor recognition sequence upstream of a minimal promoter linked to a luciferase reporter (e.g., pGL4.26)
  • using a "one-hybrid" system in which a GAL4 Binding Domain: GAL4 Upstream Activation Sequence (UAS) interaction is combined with a specific nuclear receptor ligand binding domain to modulate transcriptional activation.
  • The first method allows you to follow activation of a specific nuclear receptor pathway, but you need prior knowledge of the nuclear receptor and the nuclear response elements.
  • The "one-hybrid" method is useful if you don't know the response element for a nuclear receptor.
  • The ligand binding domain is responsible for receptor dimerization, corepressor and coactivator binding.
  • You can screen for antagonists, corepressors, coactivators, etc. in any cell line you choose.

Here's how it works:

  • The key is an optimized pGL4 vector containing 9 repeats of the GAL4 UAS, a minimal promoter and the luc2P luciferase gene (pGL4.35).
  • The preferred vector for creating the GAL4 binding domain fusion is the pFN26 (BIND) hRluc-neo Flexi® Vector.
  • Transfect your cells with both vectors, culture and assay.
  • Two prepared pFN26A vectors containing the estrogen receptor ligand binding domain (pBIND-Er∝) and the glucocorticoid receptor ligand binding domain (pBIND-GR) are available.

Confirming Coactivator Interactions with the Vitamin D Receptor Using a One-Hybrid Approach

The authors of this paper used vectors from the CheckMate™ Mammalian Two-Hybrid System to show that he peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) is a coactivator of the vitamin D receptor.

Savkur, R.S. et al. (2005) Mol. Pharmacol. 68, 511–17.

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Constructs DMSO VitD3 Fold Increase Luc Activity
Gal4BD-VDR + pG5-luc + - 1
- + 5
Gal4BD-VDR + PGC-1α + pG5-luc + - 1
- + up to 500
Gal4BD-VDR(ΔAF-2) + PGC-1α + pG5-luc + - 1
- + 1

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