Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

Ogrodzinski, Lydia; Platt, Simon; Goulding, Joelle; Alexander, Cameron; Farr, Tracy D.; Woolard, Jeanette; Hill, Stephen J.; Kilpatrick, Laura E.

Abstract

E-selectin is expressed on endothelial cells in response to inflammatory cytokines and mediates leukocyte rolling and extravasation. However, studies have been hampered by lack of experimental approaches to monitor expression in real time in living cells. Here, NanoLuc Binary Technology (NanoBiT) in conjunction with CRISPR-Cas9 genome editing was used to tag endogenous E-selectin in human umbilical vein endothelial cells (HUVECs) with the 11 amino acid nanoluciferase fragment HiBiT. Addition of the membrane-impermeable complementary fragment LgBiT allowed detection of cell surface expression. This allowed the effect of inflammatory mediators on E-selectin expression to be monitored in real time in living endothelial cells. NanoBiT combined with CRISPR-Cas9 gene editing allows sensitive monitoring of real-time changes in cell surface expression of E-selectin and offers a powerful tool for future drug discovery efforts aimed at this important inflammatory protein.

Citation

Ogrodzinski, L., Platt, S., Goulding, J., Alexander, C., Farr, T. D., Woolard, J., Hill, S. J., & Kilpatrick, L. E. (2023). Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells. iScience, 26(7), Article 107232. https://doi.org/10.1016/j.isci.2023.107232

• Journal Article Type: Article
• Acceptance Date: Jun 23, 2023
• Online Publication Date: Jun 30, 2023
• Publication Date: Jul 21, 2023
• Deposit Date: Jul 4, 2023
• Publicly Available Date: Jul 5, 2023
• Journal: iScience
• Electronic ISSN: 2589-0042
• Publisher: Cell Press
• Peer Reviewed: Peer Reviewed
• Volume: 26
• Issue: 7
• Article Number: 107232
• DOI: https://doi.org/10.1016/j.isci.2023.107232
• Keywords: NanoLuc, NanoBiT, E-selectin, Inflammatory mediators, TNFα, real-time kinetics
• Public URL: https://nottingham-repository.worktribe.com/output/22714500
• Publisher URL: https://www.cell.com/iscience/fulltext/S2589-0042(23)01309-3?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2589004223013093%3Fshowall%3Dtrue