【摘要】 目的比较不同启动子的慢病毒转导细胞后,在不同细胞系中驱动绿色荧光蛋白表达的效率高低。方法采用3种不同启动子的转移质粒,与包装质粒共转染293T细胞,转染60 h后,收集慢病毒上清。用等量的三种慢病毒液转导5种细胞系(293A、MOLT-4、PC3、DU145及RM1),72 h后,荧光显微镜下观察转导效果;流式细胞仪计数转导效率。结果不同启动子(Ubiquitin,EF1α,CMV)在5种细胞系中驱动绿色荧光蛋白的表达及转导效率不同。在293A和PC3细胞中,CMV为最强启动子,转导率分别为(94.83±2.87)%和(20.90±3.15)%;但在MOLT-4和DU145细胞中,EF1α为最强启动子,转导率分别为(74.27±2.14)%和(25.13±4.95)%;在RM1细胞中,Ubiquitin为最强启动子,转导率为(16.77±0.38)%。结论在慢病毒载体介导基因表达研究中,要考虑选取合理的细胞和启动子以获得高效的转导效率。更多还原

【Abstract】 Objective To compare the GFP expression efficiency of transduced cells driven by lentivirus with different promoters.Methods 293 T cells were co-transfected with packaging plasmids and three kinds of transfer plasmids.Then the supernatant was collected after 60 hours.5 cell lines(293A,MOLT-4,PC3,DU145 and RMl) were transduced by equal load of these lentivirus.72 hours later,GFP expression was observed with florescence microscope and transduction efficency was counted by FASC.Results GFP expression and transduction efficiency among 5 cell lines were different.Among 293 A and PC3 cells,CMV promoter was the strongest one and transduction efficiency can achieve(94.83 ± 2.87)%and(20.90 ± 3.15)%,respectively.But among MOLT-4 and DU145 cells,EFla promoter was the strongest one and transduction efficiency can achieve(74.27 ± 2.14)%and(25.13 ± 4.95)%,respectively.In RMl cells,Ubiquitin promoter was the strongest one and it can achieve(16.77 ± 0.38)%.Conclusion In the study of gene expression mediated by lentivirus,proper cell lines and promoters should be selected to obtain high efficiency.更多还原