利用 CRISPR/Cas9 构建新型基因敲除大鼠模型的特性研究。

Characterization of a Novel Knockout Rat Model Constructed by CRISPR/Cas9.

机构信息

Changning Maternity and Infant Health Hospital (D.S., J.Lu, Y.Z., J.Liu, B.Y., Y.G., X.W.), Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences (D.S, J.Lu, Y.Z., J.Liu, X.W.), East China Normal University, Shanghai, People's Republic of China and Department of Cardiology, Central Hospital of Shanghai Putuo District (Z.L.), Shanghai University of Traditional Chinese Medicine, Shanghai, People's Republic of China.

出版信息

Drug Metab Dispos. 2021 Aug;49(8):638-647. doi: 10.1124/dmd.121.000403. Epub 2021 Jun 1.

DOI:10.1124/dmd.121.000403

PMID:34074728

Abstract

CYP1A2, as one of the most important cytochrome P450 isoforms, is involved in the biotransformation of many important endogenous and exogenous substances. CYP1A2 also plays an important role in the development of many diseases because it is involved in the biotransformation of precancerous substances and poisons. Although the generation of knockout (KO) mouse model has been reported, there are still no relevant rat models for the study of CYP1A2-mediated pharmacokinetics and diseases. In this report, KO rat model was established successfully by CRISPR/Cas9 without any detectable off-target effect. Compared with wild-type rats, this model showed a loss of CYP1A2 protein expression in the liver. The results of pharmacokinetics in vivo and incubation in vitro of specific substrates of CYP1A2 confirmed the lack of function of CYP1A2 in KO rats. In further studies of potential compensatory effects, we found that was significantly upregulated, and , , and liver X receptor were downregulated in KO rats. In addition, KO rats exhibited a significant increase in serum cholesterol and free testosterone accompanied by mild liver damage and lipid deposition, suggesting that CYP1A2 deficiency affects lipid metabolism and liver function to a certain extent. In summary, we successfully constructed the KO rat model, which provides a useful tool for studying the metabolic function and physiologic function of CYP1A2. SIGNIFICANCE STATEMENT: Human CYP1A2 not only metabolizes clinical drugs and pollutants but also mediates the biotransformation of endogenous substances and plays an important role in the development of many diseases. However, there are no relevant CYP1A2 rat models for the research of pharmacokinetics and diseases. This study successfully established CYP1A2 knockout rat model by using CRISPR/Cas9. This rat model provides a powerful tool to study the function of CYP1A2 in drug metabolism and diseases.

摘要

CYP1A2 作为最重要的细胞色素 P450 同工酶之一，参与许多重要内源性和外源性物质的生物转化。CYP1A2 还在许多疾病的发展中发挥重要作用，因为它参与了致癌物质和毒物的生物转化。尽管已经报道了 CYP1A2 基因敲除 (KO) 小鼠模型的建立，但目前仍没有用于研究 CYP1A2 介导的药代动力学和疾病的相关大鼠模型。在本报告中，我们成功地通过 CRISPR/Cas9 技术建立了 CYP1A2 基因敲除大鼠模型，且没有检测到任何脱靶效应。与野生型大鼠相比，该模型肝脏中 CYP1A2 蛋白表达缺失。体内药代动力学和体外特异性 CYP1A2 底物孵育的结果证实了 KO 大鼠 CYP1A2 功能缺失。在进一步研究潜在的代偿效应时，我们发现 KO 大鼠中显著上调，而、、和肝 X 受体下调。此外，KO 大鼠血清胆固醇和游离睾酮显著升高，伴有轻度肝损伤和脂质沉积，表明 CYP1A2 缺乏会在一定程度上影响脂质代谢和肝功能。总之，我们成功构建了 CYP1A2 基因敲除大鼠模型，为研究 CYP1A2 的代谢功能和生理功能提供了有用的工具。

意义陈述

人 CYP1A2 不仅代谢临床药物和污染物，还介导内源性物质的生物转化，在许多疾病的发生发展中发挥重要作用。然而，目前尚无用于研究药代动力学和疾病的相关 CYP1A2 大鼠模型。本研究通过 CRISPR/Cas9 成功构建了 CYP1A2 基因敲除大鼠模型。该大鼠模型为研究 CYP1A2 在药物代谢和疾病中的功能提供了有力工具。