title: Complete Knockout of Endogenous Mdr1 (Abcb1) in MDCK Cells by CRISPR-Cas9.
abstract: 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.
authors: Department of Pharmacy, Uppsala University, Uppsala, Sweden.
authors: Ivailo
authors: I
authors: Simoff
authors: Department of Pharmacy, Uppsala University, Uppsala, Sweden.
authors: Maria
authors: M
authors: Karlgren
authors: Department of Pharmacy, Uppsala University, Uppsala, Sweden; Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Science for Life Laboratory, Uppsala, Sweden.
authors: Maria
authors: M
authors: Backlund
authors: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors: Anne-Christine
authors: AC
authors: Lindström
authors: Department of Pharmacy, Uppsala University, Uppsala, Sweden.
authors: Fabienne Z
authors: FZ
authors: Gaugaz
authors: Department of Pharmacy, Uppsala University, Uppsala, Sweden.
authors: Pär
authors: P
authors: Matsson
authors: Department of Pharmacy, Uppsala University, Uppsala, Sweden; Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Science for Life Laboratory, Uppsala, Sweden. Electronic address: per.artursson@farmaci.uu.se.
authors: Per
authors: P
authors: Artursson
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004285:Dogs; D055786:Gene Knockout Techniques / Q000379:methods*; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1
keywords: ABC transporters; CRISPR-Cas9; MDCK cells; P-glycoprotein; drug transport; efflux pumps; genomics; membrane transporter; permeability; proteomics