CRISPR/Cas9 System-mediated CSN2 Gene Knock-out for Preparation of Goat Fetal Fibroblasts Mutation Cells

doi:10.13560/j.cnki.biotech.bull.1985.2017-0218

Abstract

Abstract: To study the effects of beta casein（CSN2）on lactation performance of goat,the CSN2 gene of goat fetal fibroblast（GFFs）were knocked out by CRISPR/Cas9 system for providing the nuclear donors for the construction of transgenic animal. Five sgRNA targeting sites（T1,T2,T3,E1,and E2）were designed for CSN2 targeting at second and eighth exons and ligated to vector PX330-eGFP,thus PX330-eGFP-sgRNA expression vector was constructed. The knockout efficiency was evaluated by T7E I enzyme digestion experiment. Moreover,the sgRNA with the high knock-out efficiency of eighth exons was selected for constructing the PX330-Neo-sgRNA expression vector. Then the PX330-eGFP-sgRNA T and PX330-eGFP-sgRNA E combined with PX330-Neo-sgRNA E were transfected into cells,and mutant cells with CSN2 knock-out were obtained by flow sorting and G418 screening. The expression of CSN2 in the transfected cell was measured by Western blot. Sequencing results showed that the sgRNA was correctly ligated to the PX330 expression vector,T7E I digestion experiments showed that the knock-out efficiency of sgRNA T1 and sgRNA E2 was 34.9% and 25.9%,respectively. The proportion of CSN2 knock-out cell in eGFP+Neo transfection group was significantly higher than that in eGFP+eGFP transfection group（P<0.05）. In summary,we draw the conclusion that CRISPR/Cas9 system would be efficiently applied to the gene editing of GFFS and the targeting mutation cell will be of the potential resourse of nuclear donor in generating CSN2 gene knock-out goat.

Key words: milk goat, CRISPR/Cas9 system, knock-out, β-casein

JIA Qi-peng,SHEN Pei-lei,ZHANG huan,ZHANG Yong. CRISPR/Cas9 System-mediated CSN2 Gene Knock-out for Preparation of Goat Fetal Fibroblasts Mutation Cells[J]. Biotechnology Bulletin, 2017, 33(9): 131-138.

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