Kent, T.C. and Thompson, K.S. and Naylor, L.H. (2005) Development of a generic dual-reporter gene assay for screening G-protein-coupled receptors. J Biomol Screen, 10 (5). pp. 437-446. ISSN 1087-0571 .
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Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screening multiple GPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stable HEK293 cell lines were generated expressing either a firefly (Photinus) luciferase gene under the control of multiple cAMP-response elements (CREs) or a Renilla luciferase gene under the control of multiple 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligand binding activity at both Galphas-and Galphaq-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activity was assessed at Galphai/o-coupled receptors in combination with either Galphas-or Galphaq-coupled receptors. The dual-reporter gene assay was shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Z' factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.
|Additional information:||1087-0571 (Print) Journal Article Research Support, Non-U.S. Gov't|
|Uncontrolled keywords:||Animals Biotechnology/*methods Cell Line Dose-Response Relationship, Drug Drug Evaluation, Preclinical/*methods Fireflies Gene Library *Genes, Reporter *Genetic Techniques Genetic Vectors Green Fluorescent Proteins/chemistry Humans Ligands Luciferases/metabolism Receptors, Cell Surface/metabolism Receptors, Dopamine D2/chemistry Receptors, G-Protein-Coupled/genetics/*metabolism Response Elements Tetradecanoylphorbol Acetate/chemistry Transfection|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences > Cell & Developmental Biology Group|
|Depositing User:||Sue Davies|
|Date Deposited:||05 Sep 2008 11:17|
|Last Modified:||14 Jan 2010 14:25|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/6724 (The current URI for this page, for reference purposes)|
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