携带xy1E基因的穿梭载体诱变分析系统的建立及应用

Establishment and appliment of a mutation analysis system of shuttle vector carrying xyIE gene

在ＥＢ病素的基础上，以邻苯二酚氧化酶基因（ｘｙ１Ｅ）为靶基因，新霉素抗性基因（ｎｅｏｒ）为选择基因构建了穿梭质粒ｐＥＳｎｘ１及ｐＥＳｎｘ２。ｐＥＳｎｘ１转染人羊膜细胞ＦＬ，得到一株带有完整的自主复制型ｐＥＳｎｘ１质粒的Ｇ４１８抗性细胞株ＦＥＳ１．７。该细胞株平均每细胞含有质粒２６个拷贝，且在８个月的传代培养中保持稳定。ｘｙ１Ｅ在ＦＥＳ１．７细胞中的自发突变率仅４．３×１０（－５），为一高灵敏度的诱变分析系统。应用该系统对ＥＭＳ诱导的ＦＥＳ１．７细胞突变型进行分析，发现突变类型主要为缺失突变。

Two shuttle vector plasmids (pESnx1 and pESnx2),which cerrying a catechol 2, 3-dioxygenase gene(xy1E)as a target gene and a neorgene as a selected gene are constructed. They are based on Epstein-Barr Virus (EBV)and can replicate autonomously as a plasmid in both bacteria and mammalian cells. The plasmids are introduced into human amnion cell line (FL). After selected with G418,FES1. 7 was obtained which carries 26 copies of pESnx1 plasmid per cell. It was proved that the cell line FES1. 7 is stable in at least eight months passage in culture. The spontaneous mutation frequency of xy1E locus of pESnx1 in FES1. 7 cell line is 4. 3 ×10-5. Therefore,this is suitable for establishing a mutation analysis system. By using this system a deletion mutation have been detected in the FES1. 7 cells by treatment with EMS for 24 hours.