Liu Chan, Chen Chun-Yan, Shang Qian-Hui, Liu Juan
Institute of Clinical Medicine & Institute of Cardiovascular Disease & Hypertension Laboratory, the Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China.
Cardiovascular Department, the Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China.
Sheng Li Xue Bao. 2019 Aug 25;71(4):588-596.
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.
本研究的目的是利用CRISPR/Cas9基因靶向技术建立Ace2（血管紧张素转换酶2）基因敲除小鼠模型。用单向导RNA（gRNA）引物构建靶向Ace2基因敲除的载体，然后将转录的gRNA/Cas9 mRNA显微注射到小鼠受精卵中。通过PCR和基因测序检测并鉴定小鼠中Ace2基因外显子3至18的缺失情况。对Ace2基因敲除小鼠进行繁殖和交配。通过蛋白质免疫印迹法（Western blot）和实时定量逆转录聚合酶链反应（qRT-PCR）检测F3代基因敲除雄性小鼠中Ace2蛋白和mRNA的表达。成功构建了gRNA表达载体并在体外转录，将活性gRNA和Cas9 mRNA直接注射到受精卵中。通过PCR和基因测序证实了作为F0代的6只阳性奠基小鼠中Ace2基因外显子3至18的缺失。将6只奠基小鼠与野生型小鼠交配，然后将获得的F1代进行交配并产生F2代。选择F2代的雌性阳性小鼠与野生型小鼠交配并产生F3代Ace2小鼠。在这些Ace2小鼠的组织中未检测到Ace2 mRNA和蛋白。总之，利用CRISPR/Cas9技术成功建立了Ace2基因缺陷的小鼠模型，这将为今后对Ace2的研究奠定基础。
