Establishment of endogenous canine MUC1 knock-out MDCKII cells using CRISPR-Cas9 and evaluation of drug permeation

Abstract

Most orally administered drugs are absorbed by simple diffusion across the intestinal epithelium. Monolayers of MDCKII cells and parallel artificial membrane permeability assay are widely used to evaluate simple diffusion as an in vitro model; however, these models do not account for the contribution of mucus glycoprotein, which may play a significant role in drug permeation. We focused on the role of MUC1, a membrane-bound mucin that is found on the luminal surface of the gastrointestinal epithelium, in the simple diffusion of lipophilic drugs. We generated endogenous canine Mdr1 (cMdr1) and Muc1 (cMuc1) knock-out MDCKII cells by genomic editing and evaluated the effect of cMuc1 on the simple diffusion of various drugs. The absence of cMuc1 significantly increased the membrane permeation of lipophilic drugs, such as griseofulvin as well as paclitaxel and rhodamine 123, substrates of the MDR1 efflux transporter, which suggests that cMuc1 is one of the key factors that modulate the membrane permeation of these drugs. Taken together, we successfully established MDCKII cell lines with a complete knock-out of endogenous cMuc1 and cMdr1 expressions. This provides a novel in vitro model system for studying the mechanisms underlying drug absorption and transport, with potential applications for drug development.