| Genetic recombination as a reporter for screening steroid receptor agonists and antagonists. | |
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MedLine Citation:
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PMID: 9527854 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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J M Dias; N F Go; C P Hart; L C Mattheakis |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Analytical biochemistry Volume: 258 ISSN: 0003-2697 ISO Abbreviation: Anal. Biochem. Publication Date: 1998 Apr |
Date Detail:
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Created Date: 1998-04-28 Completed Date: 1998-04-28 Revised Date: 2006-08-24 |
Medline Journal Info:
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Nlm Unique ID: 0370535 Medline TA: Anal Biochem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 96-102 Citation Subset: IM |
Copyright Information:
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Copyright 1998 Academic Press. |
Affiliation:
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Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, California 94304, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Animals Base Sequence CHO Cells Cricetinae DNA, Recombinant / genetics Genes, Reporter* Hormone Antagonists / metabolism Humans Integrases / genetics Kinetics Ligands Luciferases / genetics Mice Mifepristone / metabolism Molecular Sequence Data Plasmids / genetics Receptors, Glucocorticoid / genetics, metabolism Receptors, Steroid / agonists*, antagonists & inhibitors* Recombination, Genetic* Transfection Viral Proteins* |
| Chemical | |
Reg. No./Substance:
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0/DNA, Recombinant; 0/Hormone Antagonists; 0/Ligands; 0/Receptors, Glucocorticoid; 0/Receptors, Steroid; 0/Viral Proteins; 84371-65-3/Mifepristone; EC 1.13.12.-/Luciferases; EC 2.7.7.-/Cre recombinase; EC 2.7.7.-/Integrases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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