摘要
目的探讨利用荧光激活细胞分选技术获得有效重组逆转录病毒包装细胞系的方法。方法用脂质体介导pLEGFP转染PA317细胞,用荧光显微镜观察转染结果;依据EGFP荧光利用流式细胞仪的分选技术获得多克隆和单克隆源性的包装细胞,并利用PCR、RT-PCR对其鉴定;以NIH3T3为靶细胞,对其滴度进行测定。结果荧光显微镜显示用脂质体介导的方法成功的转染PA317细胞,在4次连续流式细胞仪分选后得到了稳定表达的多克隆和单克隆源性的包装细胞。PCR和RT-PCR分别从多克隆和单克隆源性的包装细胞细胞基因组DNA以及多克隆和部分(6/8)单克隆源性的包装细胞培养上清中的重组逆转录病毒RNA中扩增出了插入的EGFP片段。滴度测定显示得到的包装细胞能够产生有效的病毒滴度。结论荧光激活细胞分选技术是获得有效病毒滴度的包装细胞的有效方法。
Objective To develop a method for acquisition of efficient and stable retroviral packaging cells on the basis of fluorescence-activated cell sorting (FACS) technique. Methods PA317 cells were transfected by the recombinant retroviral vector pLEGFP via liposome, and the result of transfection was examined using fluorescent microscope. Polyclones and monoclone of the packaging cells were obtained with FACS and identified by PCR and reverse transcriptional PCR (RT-PCR). NIH3T3 cells were used to determine the virus titer in the supernatant. Results Examination under fluorescence microscope confirmed the success of cell transfection with the retrovirus, and polyclonal and monoclonal cells with efficient and stable expression were obtained after FACS. The inserted EGFP gene fragment could be amplified by PCR from the genomic DNA of the polyclonal and monoclonal cells and by RT-PCR from the retrovirus RNA in the supernatant of monoclonal cell culture and some of the monoclonal cell cultures (6/8). Determination of the virus titer in the cell culture supernatant showed efficient viral production by the cells. Conclusion FACS is an efficient method for obtaining stable retroviral packaging cells.
出处
《第一军医大学学报》
CAS
CSCD
北大核心
2005年第1期30-32,36,共4页
Journal of First Military Medical University
基金
国家自然科学基金(30400163
30371252)~~
