血管紧张素Ⅱ2型受体基因敲除小鼠的构建、繁育和基因型鉴定

Breeding,building and genotyping identification of AngiotensinⅡtype 2 receptor knockout mice

目的利用CRISPR/Cas9技术构建血管紧张素Ⅱ2型受体(AngiotensinⅡtype 2 receptors,Agtr2)基因敲除(Agtr2^(-/-))小鼠,分析Agtr2^(-/-)小鼠的繁育和基因型。方法与江苏集萃药康公司联合设计执行CRISPR/Cas9技术,获得Agtr2^(-/-)F0代小鼠,通过聚合酶链式反应(PCR)对鼠尾基因型进行鉴定。F1代Agtr2^(-/-)小鼠由F0代Agtr2^(-/-)小鼠性成熟后与同窝野生型小鼠交配而得。PCR鉴定结果的可靠性则通过Western blot从蛋白水平上加以验证。结果利用CRISPR/Cas9技术成功构建了Agtr2^(-/-)小鼠并对所获小鼠进行繁育和基因型鉴定,得到了Agtr2+/+、Agtr2+/-、Agtr2^(-/-)3种基因型稳定的Agtr2基因小鼠。Western blot检测结果证实Agtr2^(-/-)小鼠心脏、脾脏、胸腺、肝脏及肾脏组织中几乎不表达Agtr2蛋白。结论成功构建了Agtr2^(-/-)小鼠模型,并通过可靠的鉴定方法和合适的繁育手段得到纯合Agtr2^(-/-)小鼠。

Objective To analyze the breeding and genotype of Agtr2^(-/-)mice by constructing AngiotensinⅡtype 2 receptor(Agtr2^(-/-))mice with CRISPR/Cas9 technique.Methods Agtr2^(-/-)F0 generation mice were obtained by using CRISPR/Cas9 gene targeted knockout technology.The genotypes of mouse tail were identified by PCR.Agtr2^(-/-)mice of F1 generation were procured from F0 generation Agtr2^(-/-)mice mating with the same litter wild-type mice after sexual maturity.Western blot was used to check on the dependability of PCR identification results from protein levels.Results The Agtr2^(-/-)mice were successfully constructed by CRISPR/Cas9 technology.The obtained mice were bred and genotypes were identified.Agtr2+/+,Agtr2+/-,and Agtr2^(-/-)stable Agtr2 gene mice were obtained.Western blot results confirmed that Agtr2 protein was almost not expressed in the heart,spleen,thymus,liver and kidney of Agtr2^(-/-)mice.Conclusion The Agtr2^(-/-)mouse model was successfully constructed through CRISPR/Cas9 technology,and the homozygous Agtr2^(-/-)mice were obtained through reliable identification methods and appropriate breeding methods.