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Dong, Shuyun; Rogan, Sarah C; Roth, Bryan L
Nature protocols, 03/2010, Letnik: 5, Številka: 3Journal Article
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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Leto | Faktor vpliva | Izdaja | Kategorija | Razvrstitev | ||||
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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Vir: Osebne bibliografije
in: SICRIS
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