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应用CRISPR/Cas9技术制备Ace2基因敲除小鼠模型及基因型的鉴定

刘婵¹, 陈春艳^1,2, 商黔惠^1,2,*, 刘娟¹

¹遵义医学院附属医院临床医学研究所，心血管病研究所，高血压研究室，遵义 563003；²遵义医学院附属医院心血管内科，遵义 563003

摘要

本研究旨在应用CRISPR/Cas9技术高效构建Ace2 (angiotensin-converting enzyme 2)基因敲除小鼠模型，并繁殖、鉴定及验证Ace2基因敲除小鼠。通过构建靶向敲除Ace2基因的载体，体外将Cas9 mRNA和向导RNA (guide RNA, gRNA)显微注射到小鼠受精卵中，通过PCR和TA克隆测序对小鼠Ace2基因的第3至18号外显子删除情况进行检测和鉴定，繁育Ace2基因敲除小鼠并利用qRT-PCR和Western blot方法验证获得的Ace2^−/Y小鼠主要脏器中Ace2 mRNA和蛋白表达情况。结果显示，顺利构建表达gRNA载体并体外转录，成功将有活性的gRNA和Cas9 mRNA直接注射入受精卵，获得6只阳性F0代初建鼠，PCR和基因测序鉴定表明成功删除了小鼠 Ace2 基因的第3至18号外显子；F0代鼠与野生型鼠回交，得到3只阳性F1代鼠，再与野生型鼠相交配得到的后代为F2代，在F2代中选择Ace2^−/+雌性杂合子鼠与野生型鼠交配，获得F3代Ace2^−/Y雄性纯合子小鼠。qRT-PCR和Western blot结果表明，F3代Ace2^−/Y小鼠肾脏和肺中未检测到Ace2 mRNA 和蛋白表达。本方法通过 CRISPR/Cas9技术成功制备了Ace2基因敲除小鼠模型，为进一步研究Ace2基因功能奠定了基础。

关键词： Ace2; 基因敲除小鼠; CRISPR /Cas9; 鉴定

分类号：R332; Q75

Establishment of Ace2 knockout mouse model with CRISPR/Cas9 gene targeting technology

LIU Chan¹, CHEN Chun-Yan^1,2, SHANG Qian-Hui^1,2,*, LIU Juan¹

¹Institute of Clinical Medicine & Institute of Cardiovascular Disease & Hypertension LaboratoryInstitute of Clinical Medicine & Institute of Cardiovascular Disease & Hypertension Laboratory, the Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China；²Cardiovascular Department, the Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China

Abstract

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2^−/Y mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2^−/Y mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.

Key words: Ace2; gene knockout mice; CRISPR/Cas9; identification

收稿日期：2018-10-30　　录用日期：2019-02-13

通讯作者：商黔惠　　E-mail: qhshang60@126.com

DOI: 10.13294/j.aps.2019.0046

引用本文：

刘婵, 陈春艳, 商黔惠, 刘娟. 应用CRISPR/Cas9技术制备Ace2基因敲除小鼠模型及基因型的鉴定[J]. 生理学报 2019; 71 (4): 588-596.

LIU Chan, CHEN Chun-Yan, SHANG Qian-Hui, LIU Juan. Establishment of Ace2 knockout mouse model with CRISPR/Cas9 gene targeting technology. Acta Physiol Sin 2019; 71 (4): 588-596 (in Chinese with English abstract).