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
Affiliations
Lydia Ogrodzinski
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, NG7 2UH Nottingham, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, Nottingham, UK
Simon Platt
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, NG7 2UH Nottingham, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, Nottingham, UK
Joelle Goulding
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, NG7 2UH Nottingham, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, Nottingham, UK
Cameron Alexander
Division of Molecular Therapeutics and Formulation, School of Pharmacy, Boots Building, University of Nottingham, NG7 2RD Nottingham, UK
Tracy D. Farr
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, NG7 2UH Nottingham, UK
Jeanette Woolard
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, NG7 2UH Nottingham, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, Nottingham, UK
Stephen J. Hill
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, NG7 2UH Nottingham, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, Nottingham, UK; Corresponding author
Laura E. Kilpatrick
Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, Nottingham, UK; Division of Bimolecular Science and Medicinal Chemistry, School of Pharmacy, Biodiscovery Institute, University of Nottingham, NG7 2RD Nottingham, UK; Corresponding author
Summary: 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.
