论文部分内容阅读
本研究通过慢病毒载体将红色荧光蛋白(DsRed)导入小鼠淋巴瘤EL4细胞,建立稳定高表达DsRed的EL4/DsRed细胞株,为建立携带红色荧光标记的小鼠肿瘤模型奠定基础。构建含新霉素耐药基因(neo)、内部核糖体进入位点序列(IRES)及DsRed的双顺反子自身失活型慢病毒载体。采用脂质体转染法将慢病毒三质粒包装系统共转染人胚肾细胞系293FT包装细胞,收集病毒上清,感染EL4细胞;利用G418的药物选择特性筛选感染细胞获得稳定表达DsRed的EL4细胞株,并扩大培养。结果表明:成功构建慢病毒表达质粒pXZ208-neo-IRES-DsRed,包装的重组慢病毒滴度可达106U/ml。病毒感染EL4细胞,经终浓度为600μg/ml的G418成功筛选出稳定携带DsRed的EL4/DsRed细胞株;荧光显微镜观察及流式细胞仪检测证实该细胞株长期稳定高表达DsRed。结论:通过表达DsRed的慢病毒载体感染EL4细胞,获得了稳定高表达DsRed的EL4/DsRed细胞株。
In this study, red fluorescent protein (DsRed) was introduced into mouse EL4 lymphoma cells by lentiviral vector to establish EL4 / DsRed cell line stably expressing DsRed, which laid the foundation for the establishment of a tumor model carrying red fluorescent marker in mice. To construct bicistronic self-inactivating lentivirus vector containing neomycin resistance gene (neo), internal ribosome entry site sequence (IRES) and DsRed. The lentiviral three plasmid packaging system was co-transfected into 293FT packaging cells of human embryonic kidney cell line by lipofection method, and the supernatant of virus was collected to infect EL4 cells. The infected cells were screened by drug selection of G418 to obtain EL4 stably expressing DsRed Cell lines, and expand the culture. The results showed that the recombinant lentivirus titer of pXZ208-neo-IRES-DsRed was successfully constructed and its titer was 106U / ml. EL4 cells were infected with EL4 cells and EL4 / DsRed cells stably carrying DsRed were successfully screened by G418 with a final concentration of 600 μg / ml. Fluorescence microscopy and flow cytometry confirmed that DsRed was stable and highly expressed in long-term. Conclusion: The EL4 / DsRed cell line stably expressing DsRed was obtained by infecting EL4 cells with lentiviral vector expressing DsRed.