« Previous Abstract"[Effects of mating experience and temperature on sex pheromone production of beet armyworm, Spodoptera exigua]"    Next Abstract"Corrigendum: Resisting majesty: Apis cerana, has lower antennal sensitivity and decreased attraction to queen mandibular pheromone than Apis mellifera" »

Nat Protoc

Title:		Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs
Author(s):		Dong S; Rogan SC; Roth BL;
Address:		"Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA"
Journal Title:		Nat Protoc
Year:		2010
Volume:		20100225
Issue:		3
Page Number:		561 - 573
DOI:		10.1038/nprot.2009.239
ISSN/ISBN:		1750-2799 (Electronic) 1750-2799 (Linking)
Abstract:		"G protein-coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug-designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6-8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations"
Keywords:		"Designer Drugs Directed Molecular Evolution/*methods Drug Design Humans Ligands Pheromones/genetics/metabolism Protein Engineering Receptors, G-Protein-Coupled/*genetics/*metabolism Saccharomyces cerevisiae/genetics/metabolism Signal Transduction;"
Notes:		"MedlineDong, Shuyun Rogan, Sarah C Roth, Bryan L eng U19 MH082441/MH/NIMH NIH HHS/ GM008719/GM/NIGMS NIH HHS/ MH061887/MH/NIMH NIH HHS/ R01 DA017204/DA/NIDA NIH HHS/ GM07040/GM/NIGMS NIH HHS/ MH087074/MH/NIMH NIH HHS/ MH082441/MH/NIMH NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't England 2010/03/06 Nat Protoc. 2010 Mar; 5(3):561-73. doi: 10.1038/nprot.2009.239. Epub 2010 Feb 25"