动物造模,CRISPR/Cas9技术 z-bio 2022-03-08 547

　　Objective To use CRISPR/Cas9 gene editing technology to efficiently construct Bpi gene knockout mouse model, and to continue to breed, identify and build stable genetic Bpi gene knockout mouse strains.

　　Methods Knockout targets were designed for both ends of exons 2-3 of Bpi gene in C57BL/6 mice, and high-activity gRNA (guide RNA) targets were screened. After microinjection into the fertilized eggs of C57BL/6 mice, the embryos were transferred to surrogate mother mice to obtain F0 generation mice, and gene mutation-positive offspring mice were obtained by genotype identification and DNA sequencing.

　　Results The highly active gRNA targets were screened and the in vitro transcription product sgRNA was successfully obtained; 64 fertilized eggs in good condition were obtained by microinjection together with Cas9 mRNA and successfully transplanted into 2 surrogate mothers; 22 fertilized eggs were obtained F0 generation mice, after PCR identification and DNA sequencing, a positive mouse with a single-stranded deletion of 708 bp bases was selected for amplification, and the mutation was detected in the F1 and F2 generations, and the F2 generation successfully bred homozygous small mice mouse.

　　Conclusion The Bpi gene knockout mouse model was successfully constructed, which lays a foundation for further research on the biological function of Bpi gene and its expression products.