胶质细胞源性神经营养因子受体α1定点突变体制备及其稳定转染细胞株的构建及鉴定  

Preparation of GFRα1 mutants and establishment of PC12 cell line stably expressing constructed mutants

作　　者：朱伟[1] 张勇[1] 陈哲宇[1] 王丽梅[1] 路长林[1] 何成[1] 

机构地区：[1]第二军医大学基础医学部神经生物学教研室,上海200433

出　　处：《第二军医大学学报》2004年第10期1082-1085,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金 ( 3 0 3 70 45 4) ;国家杰出青年基金 ( 3 0 3 2 5 0 2 2 ) ;国家重点基础研究规划"973"计划课题( 2 0 0 2 CB713 80 8) ;国家教育部跨世纪优秀人才培养计划 ( 2 0 0 3年 )

摘　　要：目的 :制备胶质细胞源性神经营养因子受体 α1(GFRα1)的突变体质粒 ,并建立 r GFRα1各突变体和 RET基因双转染的稳定 RCE细胞株。 方法 :通过 Fugene6脂质体转染试剂 ,将 PCR法快速制备的 pc DNA3- r GFRα1突变体质粒分别与带潮霉素 B抗性筛选标记的 pc DNA3.1- RET质粒共转染野生型 PC12细胞 ,建立 r GFRα1各突变体和 RET基因双转染的稳定PC12细胞株。经 Western印迹和细胞免疫荧光化学方法对转染入 PC12细胞中的各 r GFRα1突变体基因和 RET基因的表达进行鉴定。结果 :Western印迹和细胞免疫荧光化学方法检测均证实了所构建的细胞株中有转入基因的正确表达。结论 :成功构建了 r GFRα1各突变体基因和 RET基因同时转入表达的稳定细胞株 ,为研究 r GFRα1中关键氨基酸的位点参与胶质细胞源性神经营养因子 (GDNF)信号转导的作用奠定基础。Objective:To construct mutants of glia cell line derived neurotrophic factor receptor alpha 1 (GFRα1) and to establish PC12 cell lines stably expressing the constructed mutants. Methods: FugGENE 6 transfect reagent was used to construct stably co-transfected PC12 cell lines by the plasmids PC DNA3-rGFRα1 wild type and rGFRα1 mutants respectively with PC DNA3.1-Ret plasmid. Postive cell clones were selected with medium containing G418 and Hygromycin. The expression and location of rGFRα1 and Ret were detected by Western blot and cell immuno-flurescene test respectively.Results: Western blot and cell immuno-fluorescence test showed that rGFRα1 mutants and Ret had been expressed correctly. Conclusion: PC12 cell lines expressing either rGFRα1 or its mutants with RET have been successfully constructed,which provides basis for detecting the key single amino acid of GFRα1 involved in its biological function.

关 键 词：胶质细胞源性神经营养因子受体α1(GFRα1) RET PC12细胞 

分 类 号：Q813.1[生物学—生物工程]