Fate and biological activity of exogenous DNA sequences during serial transfections in NIH/3T3 cells.
The efficiency and accuracy of serial transfections in NIH/3T3 fibroblasts were investigated with two plasmids carrying a dominant gene. One plasmid carried the activated ras oncogene of human origin inducing morphological alteration and the oncogenic phenotype of NIH/3T3 cells. The second plasmid carried the bacterial neoR gene conferring resistance to the neomycine analogue G 418. We observed no correlation between the presence of biologically active DNAs in primary transfectants and the capacities of these DNAs to transmit the exogenous information in a second cycle of transfection. Cellular DNA of only two of 13 ras and only 1 of 3 neoR transformants could transform NIH/3T3 in a second cycle of transfections. About half of secondary transfectants, derived from those primary transfectants which did transmit the exogenous DNA, contained apparently complete exogenous sequences and transmitted it efficiently and even with the original site of integration in the host DNA in a third cycle of transfection. Exogenous DNA sequences were amplified in the majority of secondary transfectants but did not enhance biological activity in a third cycle of transfer. The exogenous DNA was found to undergo rearrangements in oncogenic transformants propagated in cell culture.
D000818:Animals; D002460:Cell Line; D004269:DNA, Bacterial / Q000378:metabolism*; D004274:DNA, Recombinant / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D005347:Fibroblasts / Q000378:metabolism; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005821:Genetic Techniques; D005839:Gentamicins / Q000494:pharmacology; D051379:Mice; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000235:genetics; D009857:Oncogenes; D010957:Plasmids; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D005839:Gentamicins; C010680:antibiotic G 418