pEGFP-C1-GnRH/TRS在HeLa细胞中的遗传稳定性

Genetic Stability of Recombinant Plasmid pEGFP-C1-GnRH/TRS in HeLa Cells

目的研究质粒pEGFP-C1-GnRH/TRS在HeLa细胞中的遗传稳定性。方法取0代及传至第10、20、30和40代的工程细胞,在有选择压力(加G418)的条件下,测定pEGFP-C1-GnRH/TRS质粒丢失率。分别提取各代次工程细胞质粒DNA进行双酶切鉴定。将经酶切鉴定正确的第40代工程细胞质粒测序,并将各代次工程细胞加压筛选后,于荧光显微镜下观察。结果连续传10、20、30和40代后,质粒丢失率均不超过2%。不同代次的工程细胞质粒DNA经BamHⅠ/EcoRⅠ双酶切,酶切图谱相同。第40代质粒的GnRH/TRS序列与原代质粒相同。第40代细胞荧光分析与原代细胞相似。结论质粒pEGFP-C1-GnRH/TRS在HeLa细胞中有较好的遗传稳定性。

Objective To study the genetic stability of recombinant plasmid pEGFP-C1-GnRH/TRS in HeLa cells. Methods Incubate recombinant HeLa cells with pEGFP-C 1 -GnRH/TRS, of passages 0,10,20,30 and 40, in G418 -containing and G418 -free media respectively,test the screened positive clones for plasmid loss rates and observe under confocal fluorescent microscope. Extract plasmids from recombinant cells of various paasages for restriction analysis, and that from recombinant ceils of passage 40 with the expected result for sequencing. Results The loss rates of recombinant plasmid pEGFP-C1 -GnRH/TRS in HeLa cells after subculture for 40 passages were not more than 2%. The restriction maps of plasmids in HeLa cells of various passages were identical. The GnRH/TRS sequence of plasmids in HeLa cells of passage 40 was consistent with that of passage 0. The confocal fluorescent micrograph of recombinant HeLa cells of passage 40 showed no significant change as compared with that of passage 0. Conclusion Recombinant plasmid pEGFP-C1-GnRH/TRS showed good genetic stability in HeLa cells.