Gata4基因H435Y突变型小鼠模型的建立及验证

Establishment and verification of a mouse model of Gata4 gene H435Y mutation

目的选择野生型C57BL/6J小鼠为研究模型,利用CRISPR/Cas9技术建立Gata4基因H435Y突变型小鼠。方法针对Gata4基因的基因序列信息设计针对Gata4基因H435Y位点的单链向导RNA(single-guideRNA,sgRNA),经活性检测后,将具有活性的sgRNA和Cas9体外转录成RNA,通过显微注射将sgRNA、Cas9 mRNA以及含有点突变序列的donor DNA片段注射到小鼠受精卵中,通过PCR和基因测序方法对Gata4基因突变进行检测及鉴定,培育Gata4基因突变小鼠并分析后代的突变情况。结果顺利构建sgRNA载体并体外转录,成功sgRNA、Cas9 mRNA以及含有点突变序列的donor DNA片段注射到受精卵中。基因测序鉴定获得4只F0代初建鼠。选取阳性F0代小鼠与野生型C57BL/6J小鼠杂交,得到F1代鼠,再相互交配获得F2代鼠。PCR显示F2代鼠Gata4基因H435Y突变,成功建立Gata4基因H435Y突变小鼠模型并传代繁育。结论通过CRISPR/Cas9技术可以成功建立Gata4基因H435Y突变小鼠动物模型。

Objective To establish a mouse model of H435 Y mutation of Gata4 gene using CRISPR/Cas9-mediated gene targeting. Methods The single-stranded guide RNA（sgRNA） specific to the H435 Y loci of Gata4 gene was designed based on the sequence of Gata4 gene. After activity assessment, the active sgRNA and Cas9 were in vitro transcribed into RNA and microinjected along with the donor DNA fragment with point mutations into fertilized mouse eggs. The microinjected eggs were transferred into pseudopregnant mice to obtain the F0 generation mice with the target Gata4 gene mutation confirmed by PCR and gene sequencing. Gata4 gene mutations in the offsprings of the F0 generation mice were analyzed. Results Gene sequencing confirmed the successful establishment of mouse models carrying H435 Y mutation of Gata4 gene in 4 of the F0 generation mice. The positive F0 generation mice were crossed with wild-type C57 BL/6 J mice to obtain the F1 generation mice,and PCR confirmed the presence of H435 Y mutations of Gata4 gene in 6 of the F1 mice. Then F2 generation mice were obtained by F1 generation matting with each other. PCR showed that H435 Y mutation of Gata4 gene in F2 mice was found, indicating the mousemodel of Gata4 gene mutation in H435 Y was established and propagated successfully. Conclusion We successfully established Gata4 gene H435 Y mutant mouse models using CRISPR/Cas9 technique.