稳定表达GFP-V12Rac1的NIH3T3细胞系的建立
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国家自然科学基金资助(30872926)


Establishment of a stable GFP-V12Rac1 NIH3T3 cell line
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    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-

    Abstract:

    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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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(自然科学版),2010,(6):741-745

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  • 收稿日期:2010-03-29
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