The Studies And Application Of A Shuttle Vector PSP189/Mammalian Vero Cell System For Mutagenicity

A SV40 based shuttle vector pSP189 and African green monkey kidney cell line (Vero) was appied , which constitute a shuttle vector /mammalian cell system to detect mutagenesis in vitro induced by animal drugs olaqiundox and quinocetone. In the study, plasmids pSP189 were transfected into vero cells by positive ion LipofectMINA2000 reagent. A fter replicating in the mammalian cells,and then picked up and returned to E.coli MBM7070 for analyzing transformants and mutatants. The spontaneous mutation frequency was 1.9×10-4 in pSP189/vero.In order to further confirm that pSP189/vero could be utilized for detection of mutagenicity ,known mutagen MNNG and B(a)P were used to treat pSP189/vero.Results showed that the mutation frequency induced by 0.32ug.ml-1 and 1.1ug.ml-1 B(a)P were about 10.5 folds and 9 folds higher than that of the background lever.The leision induced by 6.6ug.ml-1olaqiunxox included a large number of basesubstitutions,in addition to complex deletions. Point mutation frequencies for in vitro modified plasmids were dramatically increased over the spontaneous background lever.However the number of mutation induced by 1.90,3.75, 7.5, 15, 30ug.ml-1 qiuncetone had no obvious changes over the spontaneous background.Rat-liver microsomes were used to activate quinocetone for in vitro modification of the pSP189 shuttle vector.Modified plasmids were transfected into Vero cells,then recovered and transfected into E.coli MBM7070 for mutant identification.But in the reseach can not find mutations increasing over the spontaneous background lever, so qiuncetone can not: effect plasmids DNA mutate,...