Abstract: The purpose of this study was to develop Wip1-knockout swine testis (ST) cells using CRISPR/Cas9 system, which laid a foundation for furture exploring the roles of Wip1 gene in the proliferation and apoptosis of ST cells. In this study, two sgRNA (sgWip1-1 and sgWip1-2) targeting the first exon of Wip1 gene were respectively cloned into pX330-GFP and pX330-RFP vectors, and then co-transfected into ST cells (immature Sertoli cell isolated from 80-90 day-old pig embryo testes). The positive ST cells with red and green fluorescence were selected by flow cyto-metry to screen monoclonal cells. The 30 monoclonal cells were performed for genotype identification, and then the PCR products were used to implement T-A clone. In total, 29 monoclonal cells were exhibited fragment deletions, including 5 clones with monoallelic fragment deletions, 8 clones with biallelic homozygous fragment deletions, 16 clones with biallelic heterozygous fragment deletions. The fragment knockout efficiency was 96.7%. In conclusion, we efficiently obtained the Wip1-knockout ST cells. This work not only provides a cell model for subsequent researching the effects of Wip1 gene on the proliferation and apoptosis of ST cells, but also lays a good foundation for exploring the roles of Wip1 gene on the reproductive performance of boars.

Key words: CRISPR/Cas9, Wip1 gene, ST cells, pig