PIN1基因敲除的长白猪胎儿成纤维细胞系的建立

Establishment of PIN1 knockout porcine fetal fibroblast cell lines via CRISPR/Cas9 mediated gene-targeting

目的利用成簇的规律间隔短回文重复序列和相关蛋白9(clustered regular interval short palindromic repeats/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑技术敲除长白猪胎儿成纤维细胞(porcine fetal fibroblasts,PFFs)的PIN1基因,以构建PIN1基因敲除长白猪胎儿成纤维细胞系。方法对人和猪的PIN1基因进行同源性分析。利用在线工具设计2个靶向猪PIN1基因第二个外显子区的sgRNAs,并克隆至pX330骨架质粒中。将构建成功的打靶质粒和Neomycin抗性质粒共转染至PFFs中,用G418药物筛选出抗性单细胞克隆,测序鉴定其基因型并从转录和翻译水平鉴定PIN1的表达。结果同源性分析显示人和猪PIN1蛋白的氨基酸序列一致性和相似性均为98%,二者三维结构的均方根偏差(RMSD)值为0.014。获得15个PIN1基因敲除的纯合单克隆细胞系,并且证实PIN1蛋白在翻译水平被完全敲除。结论利用双sgRNAs引导的CRSIPR/Cas9系统在PFFs中高效实现PIN1基因的双等位基因敲除,成功建立PIN1基因敲除的长白猪胎儿成纤维细胞系。

ObjectiveTo knock out PIN1 gene of porcine fetal fibroblasts(PFFs)by clustered regular interval short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system in order to establish PIN1 knockout porcine fetal fibroblast cell lines.MethodsFirstly,the homology of the PIN1 gene between humans and pigs was analyzed.Secondly,2 sgRNAs targeting the second exon region of the porcine PIN1 gene were designed using online tools(http://crispor.tefor.net/)and then cloned into the pX330(addgene#42230)skeleton plasmid.Finally,the PIN1 targeting plasmid and Neomycin resistant plasmid were co-transfected into PFFs.G418 screening was used to obtain resistant single-cell colonies.Sanger sequencing,qRT-PCR,and Western blotting were adopted to determine the PIN1 genotypes and expression levels,respectively.ResultsThe bioinformatics analysis showed that the amino acid sequence consistency and similarity of human and pig PIN1 proteins were 98%,and the root-mean-square deviation(RMSD)value of their 3-dimensional structures was 0.014.Fifteen homozygous monoclonal cell lines with PIN1 gene knockout were obtained,and the PIN1 protein was disrupted entirely in those PIN1 knockout colonies at translational level.ConclusionThe PIN1 gene is efficiently knocked out in PFFs by a double sgRNAs-guided CRISPR/Cas9 system,and the porcine fetal fibroblast cell lines with PIN1 gene knocked out are successfully established.