CRISPR/Cas9系统介导敲除CSN2基因奶山羊胎儿成纤维突变细胞的制备

摘要

旨在研究 β-酪蛋白(β-casein,CSN2)对奶山羊泌乳性能的影响,利用CRISPR/Cas9系统敲除奶山羊胎儿成纤维细胞(GFFs)CSN2基因,为转基因动物模型构建提供核供体.针对CSN2第二号、八号外显子设计5个靶向CSN2基因sgRNA(T1、T2、T3、E1和E2),与PX330-eGFP载体连接,构建PX330-eGFP-sgRNA敲除表达载体,经T7E I酶切实验评估敲除效率;选择第八号外显子敲除效率最高sgRNA,构建PX330-Neo-sgRNA敲除载体,将PX330-eGFP-sgRNA T及PX330-eGFP-sgRNA E与PX330-Neo-sgRNA E组合转染细胞,经流式分选、G418药筛获得敲除CSN2基因细胞.Western blot测定转染细胞CSN2表达情况.测序结果表明各sgRNA均正确连入PX330表达载体中,T7E 1酶切实验表明sgRNA T1、sgRNA E2敲除效率最高达34.9%和25.9%;经荧光定量PCR及免疫荧光检测结果表明,eGFP+Neo转染组CSN2敲除细胞比例显著高于eGFP+eGFP转染组(P<0.05).综上表明,CRISPR/Cas9系统可有效应用于奶山羊胎儿成纤维细胞基因编辑,产生的突变细胞为制备CSN2基因敲除奶山羊提供核供体材料.%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.