摘要： 目的 基于CRISPR/Cas9技术构建α-半乳糖苷酶A(GLA) 基因敲除的巴马公猪胎儿成纤维细胞(PFFs), 为构建人类Fabry病猪模型提供实验材料。方法 利用生物信息学方法对人/猪GLA基因编码的α-Gal A的相似性进行分析, 并鉴定出猪α-Gal A的催化残基位置。通过在线工具在编码催化残基之前的外显子区设计单链向导RNA, 构建CRISPR/Cas9打靶载体。将打靶载体与抗性质粒共转染至巴马公猪胎儿PFFs, 用G418药物筛选出单克隆细胞, 并PCR测序鉴定。结果 人/猪α-Gal A的氨基酸序列一致性为81%, 相似性为89%; 三维结构的均方根偏差值为0.012。猪α-Gal A的催化残基是第174位和第235位的天冬氨酸。成功构建CRISPR/Cas9打靶载体, 并筛选出单克隆细胞,PCR测序鉴定其基因型。结论 用生物信息学方法验证了人/猪α-Gal A具有高度的相似性。利用CRISPR/Cas9技术获得了GLA基因敲除的巴马公猪PFFs细胞系, 为构建人类Fabry病猪模型奠定了基础。

α-半乳糖苷酶A基因敲除猪胎儿成纤维细胞系的建立

冷允俊, 刘晓蕊, 李琳, 王盈, 杨海元, 戴一凡

南京医科大学江苏省异种移植重点实验室, 南京 211166

收稿日期:2021-06-23出版日期:2022-08-30发布日期:2022-07-19
通讯作者:戴一凡E-mail:daiyifan@njmu.edu.cn
作者简介:冷允俊（1995-），男，硕士研究生.
基金资助:国家自然科学基金（81874144）


关键词: Fabry病, α-半乳糖苷酶A基因, α-半乳糖苷酶A, CRISPR/Cas9, 猪胎儿成纤维细胞
Abstract: Objective To supply donor cells to generate Bama miniature pig models of Fabry disease by constructing α-galactosidase A (GLA) gene-knockout porcine fetal fibroblasts(PFFs) using CRISPR/Cas9 technology. Methods The similarity of human and pig α-Gal A coded by the GLA gene was analyzed and the catalytic residues of pig α-galactosidase A were identified using bioinformatics methods. Single guide RNA targeting the exon region oriented ahead of the region coding the catalytic residue was designed using online tools. A CRISPR/Cas9 recombinant vector was then designed before the recombinant vector and drug-resistant plasmid were co-transfected into PFFs. Monoclonal cells resistant to the action of G418 aminoglycoside were obtained and sequenced. Results The identity and similarity value of the amino acid sequence of human and pig α-Gal A were 81% and 89%, respectively. The root-mean-square deviation value of the three-dimensional structure was 0.012. The catalytic residues of pig α-Gal A were aspartic acids at positions 174 and 235. The recombinant vector was constructed and monoclonal cells were successfully obtained. The cell genotypes were identified by sequencing. Conclusion Human and pig α-Gal A have a high degree of similarity. The GLA-knockout PFFs obtained could be an essential foundation for the construction of Bama miniature pig models for Fabry disease.
Key words: Fabry disease, GLA gene, α-galactosidase A, CRISPR/Cas9, porcine fetal fibroblasts
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