动物造模 z-bio 2022-08-23 361

　　Objective To construct an inhibin knockout mouse model by gene editing technology of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) and preliminary analysis of its phenotype.

　　Methods According to the base sequence of the first exon of inhibin α subunit, the recognition sequence of single-stranded guide RNA (sgRNA) was designed, and the sgRNA expression plasmid was constructed. After in vitro transcription of sgRNA and Cas9 mRNA with T7 RNA polymerase, sgRNA/Cas9 mRNA was microinjected into fertilized eggs of C57BL/6J mice, and the gene bases of inhibin α subunit in neonatal mice were detected by PCR and gene sequencing. Mutation situation. The male and female mice that were homozygous for the knockout of the F2 gene were selected, and the testis and ovary of the male mouse were taken for observation, and analyzed by paraffin section and HE staining.

　　RESULTS: A total of 12 FO generation inhibin knockout mice were obtained. The male mouse No. 8 was backcrossed with the C57BL/6J female mouse to obtain the F1 generation mice, which were then mated with each other to obtain the F2 generation mice. The proportion of genotypes in F2 generation mice conformed to Mendel's law, and there was no embryonic lethality in gene knockout mice. The testes and ovaries of F2 homozygous mice were cancerous and sterile.

　　Conclusion The inhibin knockout mouse model was successfully constructed, and the cancerous phenotype of F2 homozygous mice showed that inhibin plays an important role in the mouse reproductive system.