应用CRISPR/Cas9技术制备Vasohibin-2基因敲除小鼠模型及验证

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,sgRNA)引物序列并克隆进入pU57-T7-GDNA载体?利用T7 RNA聚合酶体外转录sgRNA和Cas9 mRNA?将体外转录的gRNA/Cas9 mRNA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sgRNA载体并体外转录,成功将有活性的sgRNA和Cas9 mRNA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?

Establishment of Vasohibin-2 knockout mouse model with CRISPR/Cas9 gene targeting technology

Objective:To establish Vasohibin-2(Vash2) knockout mouse model with CRISPR/Cas9 gene targeting technology. Methods:A selected gene sequence of Vash2 was amplified with the primers of single-guide RNA (sgRNA),and then cloned into the plasmid pUC57-T7-GDNA. The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro. Transcribed gRNA/Cas9 mRNA was microinjected into the mouse zygote. The frame shifting mutation was validated by PCR and gene sequencing. Both the (F0 and F1 generation) knockout mice were analyzed. Results:The vector expressing sgRNA were successfully built. sgRNA and Cas9 mRNA were successfully transcribed and microinjected into mouse zygote. Five positive mice as the F0 generation were identified by gene sequencing. The No.5 mouse was selected to mate with wild-type mice,then achieved F1 generation were mated and produced F2 generation. The frame-shifted of Vash2 knockout mice (F2 generation) were evaluated by PCR and mutations were stably transmitted to the next generation. Conclusion:The Vash2 knockout mouse model was successfully built by Cas9/RNAsystemgene targeting technology,and it could be an efficient tool for Vash2 study.