动物造模,CRISPR/Cas9,Bpi基因敲除 z-bo 2020-09-25 386

　　Purpose: Use CRISPR/Cas9 gene editing technology to effectively construct a Bpi gene knockout mouse model, and continue to reproduce, identify and construct a stable Bpi gene knockout mouse strain.

　　Method: Designed knockout targets at both ends of exons 2 to 3 of the Bpi gene of C57BL/6 mice, screened highly active gRNA (guideRNA) targets, and transcribed them in vitro to obtain sgRNA (smallguideRNA). Inject C57BL/6 mice with Cas9 mRNA microscopy into fertilized eggs, transfer embryos to surrogate mother mice, obtain F0 generation mice, and perform genotyping and DNA analysis on positive offspring mice with gene mutations Order obtained.

　　Result: The highly active gRNA target was screened and the in vitro transcript sgRNA was successfully obtained. Through Cas9 mRNA, 64 fertilized eggs were obtained by microinjection, which were in good condition and were successfully transplanted into two surrogate mother mice. Only 22 F0 generation mice. After PCR identification and DNA sequencing, positive mice with single-stranded deletion of 708 bp were selected for amplification. Mutations were detected in the F1 and F2 generations. The F2 generation is homozygous. Successfully bred a paired mouse.

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