表达红色荧光蛋白的小鼠淋巴瘤EIA细胞株的建立及鉴定

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迭10^6U／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ELA细胞，获得了稳定高表达DsRed的EL4／DsRed细胞株。

Establishment and Identification of Mouse Lymphoma Cell Line EIA Expressing Red Fluorescent Protein

This study was purposed to construct a lentiviral vector encoding red fluorescent protein （DsRed） and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein （IRES-DsRed） was constructed. Human embryonic kidney 293PT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EIA cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10^6 U/lift. EIA cells were transfected by the viral solution efficiently. The transfected EIA cells expressing DsRed survived in the final concentration 600 μg/ml of CJ418. The expression of DsRed in the transfected EIA cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.