Genetic recombination as a reporter for screening steroid receptor agonists and antagonists.

Autor: Dias JM; Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, California 94304, USA., Go NF, Hart CP, Mattheakis LC
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 1998 Apr 10; Vol. 258 (1), pp. 96-102.
DOI: 10.1006/abio.1998.2583
Abstrakt: Reporter cell lines are often used for high throughput screening of chemical libraries to identify new receptor ligands. Here we show how Cre recombinase can be used in mammalian cells to screen for steroid receptor ligands. A translational fusion of Cre recombinase and the ligand binding domain of the human glucocorticoid receptor was transfected into mammalian cells with a loxP/luciferase reporter gene. The recombinase function of the fusion is dependent on ligand binding to the receptor, and Cre-mediated recombination results in constitutive expression of luciferase from the reporter gene. A stable transfected clone was 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 the receptor antagonist RU486. We also 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 well was sufficient to measure a dose response to ligand.
(Copyright 1998 Academic Press.)
Databáze: MEDLINE
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