Page 45 - 南京医科大学学报自然科学版

第42卷第7期 南京医科大学学报（自然科学版）
2022年7月 Journal of Nanjing Medical University（Natural Sciences） ·941 ·

·基础研究·

猪SALL1蛋白分子信息分析及基因敲除细胞系制备

李美双 ，赵丽华 ，李荣凤 1，2*
2
1
南京医科大学生殖医学国家重点实验室，江苏省异种移植重点实验室，江苏 南京 211166
1 2

［摘 要］ 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：利用生物信息学方法分
析人、猪、鼠 SALL1 的蛋白结构。利用在线设计软件，在猪 Sall1 基因第 1 外显子区域设计单链引导 RNA（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。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 fibroblast，PFF）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。
［关键词］ 猪；Sall1；蛋白结构；CRISPR/Cas9；猪胎儿成纤维细胞
［中图分类号］ Q319 ［文献标志码］ A ［文章编号］ 1007⁃4368（2022）07⁃941⁃07
doi：10.7655/NYDXBNS20220705

Molecular information analysis of SALL1 protein in pig and establishment of gene⁃knockout
pig cell lines

2
1
LI Meishuang ，ZHAO Lihua ，LI Rongfeng 1，2*
1 State Key Laboratory of Reproductive Medicine，Nanjing Medical University， Jiangsu Key Laboratory of
2
Xenotransplantation，Nanjing 211166，China

［Abstract］ 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 porcine fetal fibroblasts（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.
［Key words］ pig；Sall1；protein structure；CRISPR/Cas9；porcine fetal fibroblast
［J Nanjing Med Univ，2022，42（07）：941⁃947］

［基金项目］ 国家自然科学基金（31971379）
∗
通信作者（Corresponding author），E⁃mail：lirongfeng@njmu.edu.cn