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

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

doi:10.1016/j.isci.2023.107232

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

iScience. 2023 Jun 30;26(7):107232. doi: 10.1016/j.isci.2023.107232. eCollection 2023 Jul 21.

Authors

Lydia Ogrodzinski^{1

2}, Simon Platt^{1

2}, Joelle Goulding^{1

2}, Cameron Alexander³, Tracy D Farr¹, Jeanette Woolard^{1

2}, Stephen J Hill^{1

2}, Laura E Kilpatrick^{2

4}

Affiliations

¹ Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, NG7 2UH Nottingham, UK.
² Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, Nottingham, UK.
³ Division of Molecular Therapeutics and Formulation, School of Pharmacy, Boots Building, University of Nottingham, NG7 2RD Nottingham, UK.
⁴ Division of Bimolecular Science and Medicinal Chemistry, School of Pharmacy, Biodiscovery Institute, University of Nottingham, NG7 2RD Nottingham, UK.

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.

Keywords: Biotechnology; Cell biology; Molecular biology.

Abstract

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