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Nucleic Acids Research, 1987, Vol. 15, No. 2 561-573
© 1987

Articles

Plasmid mediated mutagenesis of a cellular gene in transfected eukaryotic cells

Curtis R. Brandt, Franco M. Buonaguro, James K. McDougall and Denise A. Galloway

Fred Hutchinson Cancer Research Center 1124 Columbia St, Seattle, WA 98104, USA

Received September 24, 1986. Revised December 8, 1986. Accepted December 10, 1986.

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 ({approx}1x10–7). 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 ≤1x10–7. 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.


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