文章摘要
张 敏,姚 俊,鲁雅洁,王红顺,王 盈,杨海元,魏钦俊,曹 新,戴一凡.GPRASP2基因编辑猪胎儿成纤维细胞系的建立[J].南京医科大学学报,2021,(1):4~10
GPRASP2基因编辑猪胎儿成纤维细胞系的建立
Construction of GPRASP2⁃modified porcine fetal fibroblasts
投稿时间:2019-09-18  
DOI:10.7655/NYDXBNS20210102
中文关键词: GPRASP2  CRISPR/Cas9  猪胎儿成纤维细胞  GPRASP2基因敲除细胞系
英文关键词: GPRASP2  CRISPR/Cas9  porcine fetal fibroblast  GPRASP2⁃deficient cell line
基金项目:国家自然科学基金项目(32070587);江苏省财政厅转化医学实验室平台建设项目;南京医科大学科研创新基金重大项目(2017NUMCX001)
作者单位
张 敏 南京医科大学医学遗传学系江苏 南京 211166 
姚 俊 南京医科大学医学遗传学系江苏 南京 211166 
鲁雅洁 南京医科大学医学遗传学系江苏 南京 211166 
王红顺 南京医科大学医学遗传学系江苏 南京 211166 
王 盈 南京医科大学江苏省异种移植重点实验室江苏 南京 211166 
杨海元 南京医科大学江苏省异种移植重点实验室江苏 南京 211166 
魏钦俊 南京医科大学医学遗传学系江苏 南京 211166 
曹 新 南京医科大学医学遗传学系江苏 南京 211166 
戴一凡 南京医科大学江苏省异种移植重点实验室江苏 南京 211166 
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中文摘要:
      目的:采用CRISPR/Cas9基因编辑技术构建G蛋白偶联受体相关分选蛋白2(G protein?coupled receptor associated sorting protein 2,GPRASP2)基因敲除的猪胎儿成纤维细胞(porcine fetal fibroblast,PFF),为构建GPRASP2基因敲除巴马小型猪模型提供相应的供体细胞。方法:采用生物信息学方法对人与猪GPRASP2基因进行亲缘性和同源性分析,预测人与猪GPRASP2基因编码的氨基酸序列和蛋白二级结构;设计合成靶向巴马猪GPRASP2基因编码区上游和下游的单导向RNA(single guide RNA,sgRNA),以pX330质粒为载体,构建含有Cas9骨架的重组打靶质粒,并将此重组质粒转染至PFF中,G418药物筛选阳性单克隆细胞,测序分析其基因型。结果:生物信息学分析提示人/猪GPRASP2亲缘关系相近,同源性较高,且主要功能结构域Arm2的二维和三维结构相似。构建了靶向猪GPRASP2基因的重组打靶质粒并转染PFF,通过药物筛选、基因型分析和Western blot验证,成功获得GPRASP2基因敲除单克隆PFF。结论:人/猪GPRASP2基因亲缘关系相近且高度同源;采用CRISPR/Cas9介导的基因编辑技术成功构建GPRASP2基因敲除的单克隆PFF,为建立GPRASP2基因敲除巴马小型猪模型奠定了前期基础。
英文摘要:
      Objective:To construct G protein?coupled receptor associated sorting protein 2(GPRASP2)?modified procine fetal fibroblasts(PFFs) as donor cells for the generation of GPRASP2?disrupted Bama miniature(BM)pigs. Methods:Bioinformatics methods were applied to phylogenetic and homologue analysis of human/porcine GPRASP2,and the secondary structures of human/porcine GPRASP2 proteins were predicted. Two single?guide RNAs(sgRNAs),targeting the upstream/downstream of coding sequence of the porcine GPRASP2,were designed,synthesized and ligated to pX330 plasmid. The recombinant plasmids containing Cas9 backbone were transfected into PFFs. Viable cell colonies were obtained using G418 screening and subjected to genotyping via direct PCR?based sequencing. Results:The human and porcine GPRASP2 proteins are evolutionarily closer,highly homologous,and predicted to have the similar functional Arm2 domains via 2D and 3D structure modeling. CRISPR/Cas9?sgRNA expression vectors targeting porcine GPRASP2 were constructed and transfected into PFFs. GPRASP2?deficient monoclonal PFFs were obtained by drug screening,genotypic analysis and Western blot assay. Conclusion:The human/porcine GPRASP2 proteins are evolutionarily closer and highly homologous. The GPRASP2?deficient cells were successfully constructed via CRISPR/Cas9 mediated gene editing,which provided a substantial foundation for the generation of GPRASP2?disrupted BM pigs.
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