论文部分内容阅读
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 construct an NIH3T3 cell line stably expressing GFP-V12Rac1, a GFP-fusion protein that constitutively activates Rac1. METHODS: Plasmids and lentiviral vectors expressing GFP-V12Rac1 and GFP were constructed. The NIH3T3 cell line stably expressing the target gene was obtained by lentivirus infection and flow cytometry. The function of GFP-V12Rac1 was tested by spreading experiment and the cell motility was tested by Boyden chamber migration assay. Results: NIH3T3 cell line and control cell line stably expressing GFP-V12Rac1 were established. Stably expressing GFP-V12Rac1 could promote the spreading of NIH3T3 cells. Meanwhile, the constructed cell lines had chemotaxis ability. CONCLUSION: NIH3T3 cell line stably expressing GFP-V12Rac1 can be constructed by lentiviral vector. The expression products of exogenous gene are normal and chemotactic. This cell line can be used as a reliable cellular model for studying the localization mechanism of Rac1 activity.