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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

Received: 2018-10-30　　Accepted: 2019-02-13

Corresponding author: 商黔惠　　E-mail: qhshang60@126.com

DOI: 10.13294/j.aps.2019.0046

Citing This Article：

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).