A one-arm homologous recombination approach for developing nuclear receptor assays in somatic cells.
Drug discovery is in need of technologies that enable investigators to develop cell-based assays that accurately reflect the functional consequence of small molecule intervention on biological processes. Here, we describe a strategy that uses both one-arm homologous recombination and the beta-lactamase (BLA) reporter system, a sensitive and robust transcriptional reporter for gene activation. We demonstrate that this powerful approach can be utilized for developing cell-based assays for the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in HEK293 somatic cells. Specifically, one-arm homologous recombination was used to introduce the GAL4 DNA-binding domain (GAL4DBD) in the GR and MR genomic loci such that a chimeric GAL4DBD-GR (ligand-binding domain) [GAL4DBD-GR(LBD)] and GAL4DBD-MR(LBD) transcript is produced from the strong CMV promoter in HEK293 cells previously stably transfected with the UAS(GAL4)-BLA reporter construct. Dexamethasone- and aldosterone-responding BLA-positive cells were isolated by fluorescence-activated cell sorting, and then further expanded into separate cell lines. The sensitivity and robustness of the resulting GR and MR assays are demonstrated by the fact that the addition of dexamethasone and aldosterone to the two transgenic clonal cell lines for 16 h results in high Z' values (>0.8) and EC(50) values of 1 and 0.3 nM, respectively. These assays illustrate the flexibility of this technology to generate high-performance cellular assays for nuclear receptor targets without the need for target-specific cDNA.
Qureshi
Sohail A
SA
Department of Neurology, University of Wisconsin, Madison, WI, USA.
Sanders
Pam
P
Zeh
Karin
K
Whitney
Mike
M
Pollok
Brian
B
Desai
Radhika
R
Whitney
Pamela
P
Robers
Matt
M
Hayes
Steven A
SA
D000450:Aldosterone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D006822:Hybrid Cells / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011995:Recombination, Genetic / Q000187:drug effects / Q000235:genetics*
D018160:Receptors, Cytoplasmic and Nuclear; D000450:Aldosterone