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目的 制备携带GFP基因的逆转录病毒 ,以简便、快速标记活细胞。方法 将编码GFP的cDNA片段插入逆转录病毒载体pLNCX构建重组逆转录病毒载体pLNCX GFP ,借助脂质体转染单嗜性及双嗜性逆转录病毒包装细胞 ,G418筛选抗性克隆 ,然后利用荧光显微镜选出GFP表达最高的克隆。取含病毒颗粒的上清液感染NIH3T3及多种肿瘤或血管内皮细胞。结果 重组逆转录病毒载体pLNCX GFP转染包装细胞后 ,包装细胞可表达GFP并产生GFP逆转录病毒。尽管GFP逆转录病毒对动物及人的肿瘤细胞或血管内皮细胞的感染能力各不相同 ,但由于逆转录病毒能整合进入宿主细胞基因组DNA内 ,经短期G418筛选后容易获得持续、稳定、高水平表达GFP的阳性细胞。表达GFP的肿瘤细胞仍可在同系动物体内生长并持续表达GFP。结论 携带GFP基因的逆转录病毒能简便、快速介导稳定的基因转移 ,遗传标记多种细胞 ,比表达质粒等更优越。
Objective To prepare a retrovirus carrying the GFP gene for easy and rapid labeling of viable cells. METHODS: The cDNA fragment encoding GFP was inserted into the retroviral vector pLNCX to construct the recombinant retroviral vector pLNCX GFP. The mononullophilic and amphotropic retrovirus packaging cells were transfected with the liposome and the resistant clones were screened by G418. The GFP-expressing clones were selected by microscope. Supernatants containing virus particles were infected with NIH3T3 and various tumor or vascular endothelial cells. Results After recombinant retroviral vector pLNCX GFP was transfected into packaging cells, packaging cells could express GFP and produce GFP retrovirus. Although the ability of GFP retrovirus to infect human and human tumor cells or vascular endothelial cells varies, because retroviruses can integrate into the host cell genomic DNA, sustained, stable, high levels can be easily obtained after short-term G418 selection. Positive cells expressing GFP. GFP-expressing tumor cells can still grow in syngeneic animals and continue to express GFP. Conclusion The retroviral vector carrying the GFP gene can easily and rapidly mediate stable gene transfer and genetically label a variety of cells, which is superior to expression plasmids.