携带XylE基因的穿梭质粒诱变分析系统在诱变剂检测中的应用

THE APPLICATION OF INDUCED MUTATION ANALYSIS SYS-TEM CARRYING XYLE GENE IN MUTAGEN DETECTION

以邻苯二酚氧化酶基因－ｘｙｌＥ为靶基因，以新霉素抗性基因ｎｅｏ为选择基因构建的ｐＥＳｎｘ穿梭质粒转染人羊膜细胞ＦＬ，得到一株携带有完整的自主复制型ｐＥＳｎｘ质粒的Ｇ４１８抗性细胞株ＦＥＳｌ．７。经测定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，ｐＥＳｎｘ质粒在ＦＥＳ１．７细胞内ｘｙｌＥ基因的自发突变率仅４．３×１０－５。因此由ＦＥＳ１．７细胞构成的系统可在人体细胞中进行诱变检测。该系统灵敏度高，可对低效诱变剂在人体中引起的基因突变进行检测，而且特别适合于研究人体细胞中基因突变的分子机制。利用此ＥＢＶ－ｘｙｌＥ穿梭质粒诱变分析系统我们已对ＥＭＳ进行了诱变分析，对于ＦＥｓ１．７细胞，１８９ｕＭＥＭＳ诱发的ｘｙｌＥ基因突变超过自发突变频率达２２倍。ＥＭＳ处理细胞２４小时能引起细胞内的质粒在ｘｙｌＥ基因座位上发生缺失突变。

The plasmid pESnx which carrying target gene-xylE and selected gene-neowas introduced into a human amnion cell line(FL).After selected with G418 FESl。7 cell wasobtained which carries 26 copies of pESnx per cell.It was demonstrated that the cell lineFES1.7 is stable in at least eight months passage in culture。 The frequency of xylE locus ofPESnx in FES1. 7 cell line is.3×10-5.Using this EBV-xylE shttle vector system the in-duced mutation of ethyl methanesulfonate(EMS)in cell line FES1.7 was studied.The resultshowed that l98uM EMS can induce the mutation frequency at the xylE locus with 22 timeshigher than that of the spontaneous mutation frequency.A deletion was occuried by treatingthe FES1.7 cells for 24 hours。