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pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector

by Promega
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  • Overview
  • Protocols
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  • 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-ER?) 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.
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    Sequence Information

    pFN26A (BIND) hRluc-neo Flexi® Vector GenBank® Accession Number GQ229578 and vector sequence text file.

    pBIND-ER? Vector GenBank® Accession Number GQ229579 and vector sequence text file.

    pBIND-GR Vector GenBank® Accession Number GQ229580 and vector sequence text file.

    pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector GenBank® Accession Number GQ229577 and vector sequence text file.

    pGL4.36[luc2P/MMTV/Hygro] Vector GenBank® Accession Number FJ773214 and vector sequence text file.

  • Protocols

    Complete Protocol

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    pGL4.36[luc2P/MMTV/Hygro] Vector Protocol

    PDF (168 KB)

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    pFN26A (BIND) hRluc-neo Flexi Vector Product Information

    PDF (164 KB)

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    pBIND-ER? Vector Protocol

    PDF (167 KB)

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    pBIND-GR Vector Protocol

    PDF (166 KB)

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    pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector Protocol

    PDF (155 KB)

  • Certificate of Analysis

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    Storage Conditions

    -30C TO -10C

    Use Restrictions

    For Research Use Only. Not for Use in Diagnostic Procedures.

  • Resources

    Citations