Journal
NATURE PROTOCOLS
Volume 5, Issue 3, Pages 561-573Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2009.239
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Funding
- NARSAD Distinguished Investigator Award
- National Institutes of Health
- National Institute of General Medical Sciences [GM07040, GM008719]
- National Institute of Mental Health [MH087074, MH082441, MH061887]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM008719, T32GM007040] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF MENTAL HEALTH [F30MH087074, U19MH082441, R01MH061887] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON DRUG ABUSE [R01DA017204] Funding Source: NIH RePORTER
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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.
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