Establishment of Ace2 knockout mouse model with CRISPR/Cas9 gene targeting technology.
- Author:
Chan LIU
1
;
Chun-Yan CHEN
1
;
Qian-Hui SHANG
1
;
Juan LIU
1
Author Information
1. Institute of Clinical Medicine & Institute of Cardiovascular Disease & Hypertension Laboratory, the Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China.
- Publication Type:Journal Article
- MeSH:
Animals;
CRISPR-Cas Systems;
Female;
Gene Knockout Techniques;
Gene Targeting;
Male;
Mice;
Mice, Knockout;
RNA, Guide;
genetics
- From:
Acta Physiologica Sinica
2019;71(4):588-596
- CountryChina
- Language:Chinese
-
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 mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2 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.
