| Cytochrome P450 (CYP) is a superfamily of heme containing enzymes which involve in oxidation of both endogenous and exogenous substrates including majority of prescription drug and other xenobiotics, toxins, cancerogens, steroids and fatty acids. CYPs play an important role in pharmacology or drug toxicology study. CYP2E1 is one of the most conserved xenobiotic-metabolizing enzymes in CYP superfamily and is susceptible to induction by environment and pathological state in human. Besides its drug-metabolizing capacity, CYP2E1 is responsible for hepatic toxicity caused by a population of chemicals such as alcohol and acetaminophen. Currently, several Cyp knockout mice lines have been used for associated metabolic and toxicological research. However, there are no Cyp knockout rat lines available in the world due to some technical reasons. The development of technologies in the field of molecular biology especially gene target technology, make it possible to construct gene knockout rat models. Here, CRISPR/Cas system was employed for construction of Cyp2e1 KO rat lines to investigation the in vivo functions of CYP2E1. CRISPR/Cas system is RNA-mediated adaptive immune system found in bacteria and archaea that protect against viruses and plasmids based on its characteristics of recognizing and binding target DNA sequences. In this project, guideRNA and Cas 9 mRNA were injected into cytoplasm of one-cell embryo to induce DNA double break. After F0 chimeric founder were identified and bred, homozygous KO individuals in the second generation were identified by DNA sequencing, real time-PCR and western blot assay. Pharmacokinetic study and in vitro metablic study of CYP2E1 probe substrate chlorzoxazone showed KO rats were poor to metabolize and dispose chlorzoxazone. Cyp2e1 KO rats obtained from this project provide a powerful tool to study CYP2E1 associated drug metabolism and toxicology. |
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