Establishment And Verification Of A Mouse Model Of Gata4 Gene H435Y Mutation

Objective: To establish a mouse model of H435 Y mutation of Gata4 gene using CRISPR/Cas9-mediated gene targeting.Method: The single-stranded guide RNA(sg RNA)specific to the H435 Y loci of Gata4 gene was designed based on the sequence of Gata4 gene.After activity assessment,the active sg RNA 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.Result: 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 sg RNA 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.Conclusion: We successfully established Gata4 gene H435 Y mutant mouse models using CRISPR/Cas9 technique.