Evaluation of cell-based assays for steroid nuclear receptors delivered by recombinant baculoviruses

Roy M. Katso, Janet H. Parham, Matilde Caivano, William C. Clay, J. Patrick Condreay, David W. Gray, Kathryn M. Lindley, Sarah J. Mason, Jennifer Rieger, Nicole C. Wakes, William J. Cairns, Raymond V. Merrihew

    Research output: Contribution to journalArticlepeer-review

    19 Citations (Scopus)

    Abstract

    The authors describe the use of modified baculoviruses containing mammalian expression cassettes (BacMam technology) in steroid nuclear receptor reporter assays designed for screening and profiling agonist and antagonist compounds. Baculo-viruses were constructed that express full-length human genes for mineralocorticoid receptor (MR), glucocorticoid receptor (GR), progesterone receptor A (PR-A), and progesterone receptor B (PR-B) from the cytomegalovirus immediate early promoter. A virus carrying the mouse mammary tumor virus-firefly luciferase (MMTV-Luc) cassette was generated to provide a suitable reporter construct. Feasibility studies with BacMam-MR in single-dose tests of 1000 compounds showed high correlation to the standard transfection-based assay results. Likewise, in dose-response experiments, BacMam-based assays for GR and PR-B produced potency and efficacy values similar to transfection assay results. At various receptor/reporter ratios, the BacMam assays showed good flexibility, demonstrating consistent signal-to-background (S/B) ratios and compound potencies. Increasing transduction time from 24 to 48 h provided no benefit, actually reducing overall assay performance as measured by S/B and Z' values. The BacMam technology was applied in studies of isoforms PR-A and PR-B, which showed similar responses to a series of agonists. Taken together, the results demonstrate the utility of steroid nuclear receptor BacMam constructs for compound screening procedures with high reproducibility, reduced turnaround time, and lower cost.
    Original languageEnglish
    Pages (from-to)715-24
    Number of pages10
    JournalJournal of Biomolecular Screening
    Volume10
    Issue number7
    DOIs
    Publication statusPublished - 2005

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