Category: Assay Development and Screening
GPCRs are important drug targets requiring receptor-protein interaction and trafficking studies to reveal how they function. Bioluminescence resonance energy transfer (BRET) is a versatile tool to study such interactions and trafficking. Hitherto, it is limited by the ectopic expression of labelled interaction partners. CRISPR/Cas9 genome editing overcomes the limitation by enabling endogenous expression of luciferase-labelled proteins.
HEK293 cells were edited by CRISPR/Cas9 to express the nanoluciferase(Nluc)-labeled chemokine receptor CXCR4 resulting in endogenous expression of the donor-labeled receptor. The cells have proven to provide sufficient luminescent signal to be used in nanoBRET™ experiments measuring β-arrestin recruitment after receptor activation, receptor trafficking from membrane to early endosome as well as GPCR heteromer formation.
Sensitive instrumentation was required to measure the signal derived from Nluc/CXCR4 expressed at endogenous levels as well as BRET acceptor emission. The CLARIOstar® microplate reader was employed due to its high sensitivity in monochromator-based luminescence and BRET measurements. With its broad bandwidths, the monochromator enabled the measurement of different acceptor fluorophores as well as the combined use of two acceptors in one experiment that were employed to report on cellular localization of the Nluc-labeled receptor.
Use of the revolutionary gene-editing method CRISPR/Cas9 overcomes the drawback of studying GPCRs by nanoBRET™ at ectopic expression levels. Gene-edited cell expressing the luciferase-labeled receptor at endogenous level in combination with a sensitive detector serve as a tool for comprehensive GPCR research.
Ron Earp– President, BMG LABTECH Inc., Cary NC, NC
BMG LABTECH Inc.
Cary NC, NC
Ron has a Ph.D. in Analytical Chemistry and extensive research experience in optical and label-free detection. Before coming to BMG LABTECH he worked in the pharma industry and for an instrumental design company. Currently Ron is the President of BMG LABTECH Inc. located in Cary NC.