关键词: 基因敲除；CRISPR/Cas9基因编辑技术；抑制素；癌变；C57BL/6J小鼠 

Abstract: Objective    To construct an inhibin gene knockout mouse model by using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology, and to preliminarily study the phenotypes. Methods     Single guide RNA (sgRNA) plasmids against the exon 1 of inhibin α-subunit were designed and constructed. The sgRNA and Cas9 mRNA were transcribed by T7 RNA polymerase in vitro, then mixed and microinjected into the fertilized eggs of C57BL/6J mice. PCR and gene sequencing were used to detect the mutations of inhibin α-subunit in newborn mice. Male and female mice of F2 generation with inhibin gene knockout were selected, and their testis and ovaries were taken to observe and analyze by HE staining after paraffin sections. Results    Twelve F0 generation mice with inhibin gene knockout by CRISPR/Cas9 were obtained. The No.8 male mouse was selected to mate with female C57BL/6J mice, then F1 generation mice were achieved. F1 generation mice were mated with each other to produced F2 generation, and the ratio of genotypes conforms to Mendel’s law in F2 generation. The testis and ovary of F2 generation homozygous mice were cancerous and infertile. Conclusion    The inhibin gene knockout mouse model was successfully constructed, and the cancerous phenotype of homozygous mice shows that inhibin gene plays an important role in the mouse reproductive system.

Key words: Gene knockout, CRISPR/Cas9 gene editing technology, Inhibin, Cancerization;