The use of fluorescence correlation spectroscopy to monitor cell surface β2‐adrenoceptors at low expression levels in human embryonic stem cell‐derived cardiomyocytes and fibroblasts

Abstract

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Subtype selective fluorescent ligands based on ICI 118,551 to study the human β2‐adrenoceptor in CRISPR/Cas9 genome‐edited HEK293T cells at low expression levels

“…In HEK293 T cells, the endogenous expression of β 2 ‐adrenoceptors is extremely low, and we have previously shown that β 2 ARs tagged with an N‐terminal SNAP label under endogenous promotion are not detectable by standard confocal microscopy in CRISPR/Cas9 genome‐edited HEK293 T cells (Figure ) 35 . To confirm this low level of expression, we used CRISPR/Cas9 genome editing to incorporate an N‐terminal NLuc tag to the β 2 AR under endogenous promotion as we have described previously 32 .…”

“…β 2 ARs are also known to have a propensity to form dimers and higher order oligomers 47 . To maintain the normal cellular context, we have recently used CRISPR/Cas9‐mediated homology‐directed repair to insert both luminescent and fluorescent tags onto the N‐terminus of GPCRs into the endogenous genome, where their expression is regulated by endogenous promoters 30‐32,35 . In the present manuscript, we have investigated the utility of highly selective fluorescent ligands based on the β 2 AR selective antagonist ICI 118,551 33 in combination with CRISPR/Cas9 genome editing to investigate ligand binding to endogenous β 2 ARs under the regulation of their native promoters in HEK293 T cells.…”

“…The SNAP‐β 2 AR clonal stable cell line was a gift from Dr Karolina Gherbi (University of Nottingham). CRISPR/Cas9 genome engineering of the N‐terminal region of the β 2 AR genomic locus in HEK293 T cells to incorporate either a SNAP‐ or an NLuc‐tag was performed as described previously 32,35 . Chinese Ovary Hamster (CHO) cell lines stably expressing either the β 1 AR or β 2 AR were maintained in DMEM/F12 supplemented with 10% FBS and 2 mM L‐glutamine at 37°C in 5% CO 2 .…”

“…CRISPR/Cas9 genome engineering of the N-terminal region of the β 2 AR genomic locus in HEK293 T cells to incorporate either a SNAP-or an NLuctag was performed as described previously. 32,35 Chinese Ovary Hamster (CHO) cell lines stably expressing either the β 1 AR or β 2 AR were maintained in DMEM/F12 supplemented with 10% FBS and 2 mM L-glutamine at 37°C in 5% CO 2 . The CHO cell lines were a gift from Prof. Jillian Baker (University of Nottingham).…”

“…In HEK293 T cells, the endogenous expression of β 2adrenoceptors is extremely low, and we have previously shown that β 2 ARs tagged with an N-terminal SNAP label under endogenous promotion are not detectable by standard confocal microscopy in CRISPR/Cas9 genome-edited HEK293 T cells (Figure S4). 35 To confirm this low level of expression, we used CRISPR/ Cas9 genome editing to incorporate an N-terminal NLuc tag to the β 2 AR under endogenous promotion as we have described previously. 32 Quantification of NLucβ 2 AR expression, as measured on the PHERAstar FS plate reader, showed a 20.1-fold higher expression of β 2 ARs in the exogenous stable cell line compared to the genome-edited HEK293 T cells (Figure 6A).…”