Establishment Of MiR-32-5p And MiR-30a Knockout Mouse Models

Objective To establish three different gene knockout mouse models,including miR-32-5p and miR-30 a single-knockout homozygous mouse model and miR-32-5p and miR-30 a double-knockout mouse model and to further investigate the role of miR-32-5p and mi R-30 a in the pathogenesis of calcification.This study will lay good animal models for the following research.Methods:1)By using CRISPR/Cas9 knockout technology,both the single-guide RNA of mmu-miR-32-5p(derived from mouse miR-32-5p)and mmu-miR-30a(derived from mouse miR-30a)and the mRNA of Cas9 nuclease were simultaneously injected into the fertilized eggs to obtain F0 generation gene double knockout chimeric mice at one time.2)Cross F0 generation(miR-32-5p/miR-30 a gene double-knockout chimeric mice)with wild-type(C57BL/6J)ones to get F1 generation(miR-32-5p/miR-30 a gene knockout heterozygous progeny).3)F1 generation(miR-32-5p and miR-30 a heterozygous mice)were mated to each other to get F2 and F3 generations including miR-32-5p and miR-30 a single-knockout homozygous mice and miR-32-5p/miR-30 a double-knockout mice.4)The genotype of all generation was identified by PCR after extracting DNA from mouse tails.Results1)Obtained 16 F0 generation(miR-32-5p/miR-30 a gene double-knockout chimeric mice)through CRISPR/ Cas9 gene knockout technology.2)Obtained 29 miR-32-5p/miR-30 a gene knockout heterozygous progeny in F1 generation.These mice are fertile and each production was 4 to 9.The average survival rate was 72%.3)Obtained miR-32-5p and mi R-30 a gene single knockout homozygous mice and mi R-32-5p/miR-30 a gene double knockout homozygous mice in F2 and F3 generations.ConclusionWe successfully established miR-32-5p and mi R-30 a single-knockout mouse and miR-32-5p/miR-30 a double-knockout mouse models.We got miR-32-5p and miR-30 a single-knockout mice and miR-32-5p/miR-30 a double-knockout mice in F2 and F3 generations.