Evaluation of Cell-Based Assays for Steroid Nuclear Receptors Delivered by Recombinant Baculoviruses

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. Baculoviruses 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. (Journal of Biomolecular Screening 2005:715-724)

Baculovirus Vectors for Gene Therapy

“…In vitro and in vivo gene therapy Airenne et al, 2000;Boyce and Bucher, 1996;Hofmann et al, 1995;Pieroni et al, 2001;Sarkis et al, 2000;Tani et al, 2003Cell-based assays Ames et al, 2004Jenkinson et al, 2003;Katso et al, 2005 Studies of gene function Clay et al, 2003;Pfohl et al, 2002 Studies of virology Delaney and Isom, 1998;Dwarakanath et al, 2001;Lopez et al, 2002;McCormick et al, 2002;Zhou et al, 2000 Protein production Chen et al, 2005;Ojala et al, 2004;Ramos et al, 2002 Virus vector production Cheshenko et al, 2001;McCormick et al, 2002;Poomputsa et al, 2003;Sollerbrant et al, 2001 Surface display Ernst et al, 1998;Grabherr and Ernst, 2001 Vaccine candidates Abe et al, 2003;Aoki et al, 1999;Facciabene et al, 2004;Tami et al, 2000;Yoshida et al, 2003 C. Mechanisms of Baculovirus Entry into Mammalian Cells…”

Baculovirus‐mediated gene transfer is attenuated by sodium bicarbonate

“…Thanks to the nonreplicating nature and low cytotoxicity inside mammalian cells, the large genome (≈130 kb) capable of harboring multiple genes or large inserts [4], and ease of recombinant virus construction and production, increasing efforts have been directed towards employing baculovirus for in vitro and in vivo gene delivery [5][6][7]. Further, baculovirus-mediated gene delivery into mammalian cells has been exploited for the development of cell-based assays [8], delivery of small interfering RNA [9,10], surface display of eucaryotic proteins [11], and high-level production of virus-like particles [12].…”

Bioluminescent Assays for High-Throughput Screening

“…Luciferase assays have been successfully developed for numerous GPCRs and nuclear receptors, which represent two major drug target classes. [15][16][17][18] For GPCR assays, the activity of a receptor coupled to G␣s protein is monitored by using a cyclic adenosine monophosphate (cAMP) response element (CRE) positioned upstream of a luciferase gene. Activation of the receptor causes an increase in intracellular cAMP, which in turn activates protein kinase A to phosphorylate CRE binding protein.…”