GENETIC-RECOMBINATION AS A REPORTER FOR SCREENING STEROID-RECEPTOR AGONISTS AND ANTAGONISTS

Reporter cell. lines are often used for high throughput screening of c hemical libraries to identify new receptor ligands. Here we show hom C re recombinase can be used in mammalian cells to screen for steroid re ceptor ligands, A translational fusion of Cre recombinase and the liga nd binding domain of the human glucocorticoid receptor was transfected into mammalian cells with a loxP/luciferase reporter gene. The recomb inase function of the fusion is dependent on ligand binding to the rec eptor, and Cre-mediated recombination results in constitutive expressi on of luciferase from the reporter gene, A stable transfected clone wa s isolated and used to characterize the kinetics, ligand specificity, and dose response to various receptor ligands, The Cre fusion system, unlike a transcriptional reporter using the mouse mammary tumor virus promoter, can detect binding of tile receptor antagonist RU486. We als o studied the Cre reporter in a sensitive, miniaturized, assay format using an 864-well plate and show that as few as 560 cells per assay we ll was sufficient to measure a dose response to ligand. (C) 1998 Acade mic Press.