文章摘要
王〓乐,朱一超,杜〓军,杨〓郁,胡圳圳,顾〓洛.稳定表达GFP-V12Rac1的NIH3T3细胞系的建立[J].南京医科大学学报,2010,(6):741~745
稳定表达GFP-V12Rac1的NIH3T3细胞系的建立
Establishment of a stable GFP-V12Rac1 NIH3T3 cell line
投稿时间:2010-03-29  
DOI:10.7655
中文关键词: GFP-V12Rac1  慢病毒载体  NIH3T3细胞  稳定表达  细胞迁移
英文关键词: GFP-V12Rac1  gene transfection  NIH3T3 cell  stable expression  cell migration
基金项目:国家自然科学基金资助(30872926)
作者单位
王〓乐 南京医科大学生理学系,江苏 南京〓210029 
朱一超 南京医科大学肿瘤中心,江苏 南京〓210029 
杜〓军 南京医科大学生理学系,江苏 南京〓210029 
杨〓郁 南京医科大学生理学系,江苏 南京〓210029 
胡圳圳 南京医科大学生理学系,江苏 南京〓210029 
顾〓洛 南京医科大学肿瘤中心,江苏 南京〓210029 
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中文摘要:
      目的:构建稳定表达GFP-V12Rac1(组成型活化Rac1的GFP融合蛋白)的NIH3T3细胞系?方法:构建表达GFP-V12Rac1和GFP的质粒和慢病毒载体,通过慢病毒感染和流式分选获取稳定表达目的基因的NIH3T3细胞系?通过铺展实验检测GFP-V12Rac1的功能,通过Boyden chamber迁移实验检测细胞的运动能力?结果:建立了稳定表达GFP-V12Rac1的NIH3T3细胞系及对照细胞系;稳定表达的GFP-V12Rac1可促进NIH3T3细胞的铺展,同时,构建的细胞系具备趋化运动能力?结论:用慢病毒载体可构建稳定表达GFP-V12Rac1的NIH3T3细胞系,外源基因表达产物功能正常且细胞系具备趋化能力?该细胞系可作为研究Rac1活性定位机制的可靠细胞模型?
英文摘要:
      Objective:To establish an NIH3T3 cell line that stably expresses GFP-V12Rac1(constitutively active Rac1 fused with a GFP tag). Methods:Plasmids and lentiviral vectors containing GFP-V12Rac1 and GFP were constructed. NIH3T3 was infected with lentiviral vectors,and cells stably expressing genes of interest were selected by flow cytometry. A cell spreading assay was used to confirm that exogenous GFP-V12Rac1 was of normal function;a Boyden chamber assay was used to test the motility of established cell lines. Results:Stable cell lines expressing GFP or GFP-V12Rac1 were established. GFP-V12Rac1 promoted cell spreading. Chemotaxis of established cell lines was confirmed. Conclusion:NIH3T3 cell lines stably expressing GFP-V12Rac1 were successfully established using lentiviral methods;exogenous GFP-V12Rac1 was of normal function;chemotaxis of the cell lines was confirmed. These cell lines can be used as model cells for further study of active Rac1 targeting.
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