Development and Characterization of MDR1 ( Mdr1a/b ) CRISPR/Cas9 Knockout Rat Model

• Published in: Drug metabolism and disposition Vol. 47; no. 2; pp. 71 - 79
• Main Authors: Liang, Chenmeizi, Zhao, Junfang, Lu, Jian, Zhang, Yuanjin, Ma, Xinrun, Shang, Xuyang, Li, Yongmei, Ma, Xueyun, Liu, Mingyao, Wang, Xin
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics, Inc 01.02.2019
• Subjects: Administration, Oral; Animals; ATP Binding Cassette Transporter, Subfamily B - genetics; ATP Binding Cassette Transporter, Subfamily B - metabolism; Brain; Brain - metabolism; CRISPR; CRISPR-Cas Systems - genetics; Cytochrome P-450 CYP3A - analysis; Cytochrome P-450 CYP3A - genetics; Cytochrome P-450 CYP3A - metabolism; Digoxin; Digoxin - administration & dosage; Digoxin - pharmacokinetics; Efflux; Female; Gene Knockout Techniques - methods; Genetic modification; Genomes; Glycoproteins; In vivo methods and tests; Intestine, Small - metabolism; Kidney - metabolism; Kidneys; Liver; Liver - metabolism; Male; MDR1 protein; Models, Animal; mRNA; Multidrug resistance; Nuclease; P-Glycoprotein; Pharmacology; Proteins; Rats; Rats, Sprague-Dawley; Rats, Transgenic; RNA, Messenger - analysis; RNA, Messenger - metabolism; Rodents; Small intestine; Substrates; Target detection; Tumors
• ISSN: 0090-9556; 1521-009X
• DOI: 10.1124/dmd.118.084277

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology is widely used as a tool for gene editing in rat genome site-specific engineering. Multidrug resistance 1 [MDR1 (also known as P-glycoprotein)] is a key efflux transporter that plays an important role not only in the transport of endogenous and exogenous substances, but also in tumor MDR. In this report, a novel MDR1 ( ) double-knockout (KO) rat model was generated by the CRISPR/Cas9 system without any off-target effect detected. Western blot results showed that MDR1 was completely absent in the liver, small intestine, brain, and kidney of KO rats. Further pharmacokinetic studies of digoxin, a typical substrate of MDR1, confirmed the deficiency of MDR1 in vivo. To determine the possible compensatory mechanism of (-/-) rats, the mRNA levels of the CYP3A subfamily and transporter-related genes were compared in the brain, liver, kidney, and small intestine of KO and wild-type rats. In general, a new (-/-) rat model has been successfully generated and characterized. This rat model is a useful tool for studying the function of MDR1 in drug absorption, tumor MDR, and drug target validation.