State Key Laboratory of Reproductive Medicine,Nanjing Medical University
The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)
目的：从巴马小型猪SALL1的蛋白结构出发,分析其与人SALL1蛋白的同源性,进一步制备Sall1基因敲除的巴马小型猪胎儿成纤维细胞系,为通过体细胞核移植技术获得猪肾脏发育缺陷模型提供实验材料。方法：首先利用生物信息学方法分析人、猪、鼠SALL1的蛋白结构。其次利用在线设计软件,在猪Sall1基因第一外显子区域设计sgRNA(single guide RNA,sgRNA)并将其连接至PX330质粒,构建猪Sall1基因敲除打靶载体。然后在初步转染细胞的基础上,通过Sall1基因测序分析验证PX330?sgRNA载体打靶效率,最后将打靶载体转染至原代猪胎儿成纤维细胞(Porcine fetal fibroblasts,PFFs)中,通过药物筛选获得单克隆细胞并鉴定其基因型。结果：生物信息学分析表明,相比于小鼠,猪的SALL1蛋白与人SALL1蛋白具有更高的同源性。成功构建猪Sall1基因敲除打靶载体,并获得33个单克隆细胞系,经基因测序鉴定得到16个Sall1双等位基因敲除的细胞系。结论：生物信息学方法验证了人和猪SALL1蛋白具有更高的同源性。利用CRISPR/Cas9基因编辑技术获得Sall1基因敲除细胞系，为研究Sall1基因在猪肾脏发育过程中的作用提供研究材料，并为下一步获得猪肾脏缺失模型奠定基础。
Objective: Based on the protein structure of SALL1, the homology of SALL1 protein between pig and human was analyzed. Bama miniature pig fetal fibroblast cell lines with Sall1 gene knocked-out were developed in order to providing experimental donor cells for obtaining a pig renal development deficiency model via somatic cell nuclear transfer. Methods: Firstly, bioinformatics methods were used to analyze the structural of SALL1 protein between human, pig and mouse. Secondly, sgRNA was designed on the first exon of pig Sall1 gene and connected to PX330 plasmid to construct Sall1 gene knockout target vector. Then, through the initial cells transfection, the targeting efficiency of PX330?sgRNA vector was verified by Sall1 gene sequencing analysis. Finally, the vector was transfected into primary PFFs. The monoclonal cells were obtained through drug screening and their genotypes were identified. Results: Bioinformatics analysis showed that pig SALL1 protein was more similar to human counterparts than mouse. CRISPR/Cas9 expression vector for Sall1 gene targeting was constructed and 33 monoclonal cell lines were obtained, among them 16 Sall1 biallelic mutant cell lines were identified by gene sequencing. Conclusion:Bioinformatics methods confirmed the higher homology of human and porcine SALL1 proteins. Sall1 -/- cell lines were obtained by CRISPR/Cas9 gene editing technology, which will provided research materials for studying the role of Sall1 gene on the development of pig kidney, and lay a foundation for obtaining the porcine renal development deficiency model in the next step.