稳定表达GFP-V12Rac1的NIH3T3细胞系的建立

Establishment of a stable GFP-V12Rac1 NIH3T3 cell line

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。

Objective：To establish an NIH3T3 cell line that stably expresses GFP-V12Racl （constitutively active Racl fused with a GFP tag）. Methods：Plasmids and lentiviral vectors containing GFP-V12Rael 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-V12Racl 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-V12Racl were established. GFP-V12Racl promoted cell spreading. Chemotaxis of established cell lines was confirmed. Conclusion：NIH3T3 cell lines stably expressing GFP-V12Racl were successfully established using lentiviral methods ;exogenous GFP-V12Racl 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 Racl targeting.