Plasmid mediated mutagenesis of a cellular gene in transfected eukaryotic cells.
NIH3T3 cells are widely used in transformation assays and readily take up transfected DNA. A system has been devised using NIH3T3 cells to measure the mutagenic effect of transfected DNA on recipient cell genes. NIH3T3 cells can be mutated to 6-thioguanine resistance at a frequency which suggests that at least a portion of the cells have only one functional copy of the HGPRT gene. They have a low spontaneous background mutation frequency (approximately 1 X 10(-7)). Transfection of three different plasmids into NIH3T3 cells induced 6-thioguanine resistant mutants at frequencies ranging from 3 to 11 fold above background. The mutant phenotype is stable and reversion frequencies of several mutants are less than or equal to 1 X 10(-7). Southern blot analysis of the HGPRT gene in several mutants showed that 4 of 26 mutants (15.4%) had detectable alterations in the structure of the HGPRT gene. Interestingly 3 of the 4 mutants showing rearrangements were obtained by transfection of the HSV-2 morphological transforming region.
Brandt
C R
CR
Buonaguro
F M
FM
McDougall
J K
JK
Galloway
D A
DA
D000818:Animals; D002478:Cells, Cultured; D004351:Drug Resistance; D005796:Genes; D005818:Genetic Engineering; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D013866:Thioguanine / Q000494:pharmacology
D008769:Methylnitronitrosoguanidine; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine