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目的 :利用Cas9/RNA system gene targeting技术高效构建Vash2基因敲除小鼠模型。方法 :根据Vash2基因序列,设计2对Vsah2基因的单链向导RNA(single-guide RNA,sg RNA)引物序列并克隆进入p U57-T7-GDNA载体。利用T7 RNA聚合酶体外转录sg RNA和Cas9 m RNA。将体外转录的g RNA/Cas9 m RNA显微注射入小鼠受精卵,通过PCR和基因测序对Vash2移码突变进行检测和鉴定。繁育Vash2基因敲除小鼠并分析后代突变情况。结果:顺利构建表达sg RNA载体并体外转录,成功将有活性的sg RNA和Cas9 m RNA直接注射入受精卵。基因测序鉴定获得5只F0代初建鼠。选取5号鼠与野生型鼠回交,得到F1代鼠,再相互交配得到F2代鼠。PCR显示F2代鼠Vash2基因移码突变,成功建立Vash2基因敲除小鼠模型并传代繁育。结论:通过Cas9/RNA systemgene targeting技术可以成功制备Vash2基因敲除鼠模型,是用于Vash2研究的有效工具。
OBJECTIVE: To efficiently construct Vash2 knockout mouse model by Cas9 / RNA system gene targeting technology. Methods: According to the Vash2 gene sequence, two pairs of single-guide RNA (sg RNA) primers of Vsah2 gene were designed and cloned into pU57-T7-GDNA vector. In vitro transcription of sg RNA and Cas9 m RNA using T7 RNA polymerase. In vitro transcribed g RNA / Cas9 m RNA microinjection into mouse fertilized eggs, Vash2 frameshift mutation detection and identification by PCR and gene sequencing. Vash2 knockout mice were bred and analyzed for offspring mutation. Results: The sg RNA vector was successfully constructed and transcribed in vitro. The active sg RNA and Cas9 m RNA were successfully injected into fertilized eggs. Genome sequencing identified five F0 generation mice. Select No. 5 mice and wild-type mice backcross, get F1 generation of mice, and then inter-mating F2 generation mice. PCR showed the frameshift mutation of Vash2 gene in F2 generation mice, successfully established Vash2 knockout mouse model and passaged and breeding. Conclusion: The Vash2 gene knockout mouse model can be successfully prepared by Cas9 / RNA systemgene targeting technology and is an effective tool for Vash2 research.