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Complete Knockout of Endogenous Mdr1 (Abcb1) in MDCK Cells by CRISPR-Cas9
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab.
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2016 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 105, no 2, 1017-1021 p.Article in journal (Refereed) Published
Abstract [en]

Madin-Darby canine kidney II cells transfected with one or several transport proteins are commonly used models to study drug transport. In these cells, however, endogenous transporters such as canine Mdr1/P-glycoprotein (Abcb1) complicate the interpretation of transport studies. The aim of this investigation was to establish a Madin-Darby canine kidney II cell line using CRISPR-Cas9 gene-editing technology to knock out endogenous canine Mdr1 (cMdr1) expression. CRISPR-Cas9-mediated Abcb1 homozygous disruption occurred at frequencies of around 20% and resulted in several genotypes. We selected 1 clonal cell line, cMdr1 KO Cl2, for further examination. Consistent with an on-target effect of CRISPR-Cas9 in specific regions of the endogenous canine Abcb1 gene, we obtained a cell clone with Abcb1 gene alterations and without any cMdr1 expression, as confirmed by genome sequencing and quantitative protein analysis. Functional studies of these cells, using digoxin and other prototypic MDR1 substrates, showed close to identical transport in the apical-to-basolateral and basolateral-to-apical directions, resulting in efflux ratios indistinguishable from unity.

Place, publisher, year, edition, pages
2016. Vol. 105, no 2, 1017-1021 p.
Keyword [en]
MDCK cells, CRISPR-Cas9, ABC transporters, efflux pumps, P-glycoprotein, permeability, membrane transporter, drug transport, genomics, proteomics
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-293968DOI: 10.1016/S0022-3549(15)00171-9OAI: oai:DiVA.org:uu-293968DiVA: diva2:928459
Available from: 2016-05-15 Created: 2016-05-15 Last updated: 2016-06-01

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