应用穿梭质粒建立哺乳动物细胞诱变检测系统的实验研究

Experimental Study of Use of Shuttle Vector to Develop a System of Detecting Mutagenesis in Mammalian Cells

用基于EB病毒的穿梭质粒载体pMCi5转染小鼠3T3细胞,建立一种能在DNA水平上检测化学物对哺乳动物细胞诱变作用及其机理的快速实验系统。该质粒带有诱变作用靶基因LacI,用甲基磺酸乙酯(EMS)作诱变剂进行的实验结果表明,该系统的自发突变率小于1.21×10^(-4),而EMS(300μg/ml)的诱发突变率为3.8×10^(-3)。7个突变子经EcoRI酶切证明,质粒上无大片段插入或缺失,6个EcoRI位点上没有点突变。

The paper presents a seriesof experimental studies of the use of shut-tle vector to develop a short-time mutage-nesis system of detecting gene mutation inthe mammalian cell at DNA level. The sys-tem is based on the finding that in the EBvirus the shuttle vector pMCi5 carries Laclgene. The Lacl is used as a target of indu-ced mutagenesis in mouse cells. Use pMCi5 plasmid to transform the 3T3mouse cell; expose it to a chemical muta-gen, ethyl methanesulfonate (EMS); trans-fer the treated plasmid DNA to E colistrain MC1061F'150Kan; and then plate thetransformation on a special culture mediumcontaining X-gal for rapid detection andanalysis of Lacl mutation. Results: The spontaneous mutant fre-quency of the system is less than 1.21×10^(-4).After the treatment with 300μg/ml EMS,the induced mutant frequency increases to3.8×10^(-3). No gross alteration has beendiscovered in seven Lacl mutants at DNAlevel. No point mutation has occurred inthe EcoRI site by analysis with restrictionenzyme EcoRI.