ENU-INDUCED MUTAGENESIS AT A SINGLE-A - T-BASE PAIR IN TRANSGENIC MICE CONTAINING PHI-X174

Transgenic mice containing the bacteriophage phiX174 am3 as a chromosomally integrated and recoverable marker for in vivo mutation have been produced to measure spontaneous and induced substitutions at an A:T base pair among single copies. PhiX174 was chosen for its small size (5 kb), unique sequence, and the opportunity to take advantage of previously reported in vitro data on mutation and repair; the am3 site provides sequence specificity in a reversion assay for mutation of an A:T base pair. Inbred C57Bl/6 mice have been made homozygous for approximately 100 copies of the the phage sequence without any apparent detrimental effects on the homozygous individuals. Recoveries of phage from mouse tissues are in the range of 1-5 X 10(7) PFU per mug mouse DNA; both recovery and mutation are independent of endogenous CpG methylation. Background mutation frequencies are 2-4 X 10(-7) among phage recovered from liver, brain, spleen, and kidney. Adult mice were treated with 200 mg/kg N-ethyl-N-nitrosourea, and phage were recovered at 2 and 14 days after treatment. At 2 days after treatment we observed a slight increase only among phage isolated from the brain of one mouse out of four. At 14 days after ENU treatment, there were significant increases in mutation frequencies among phage recovered from the liver (6 X) and spleen (10 X). These results demonstrate (1) response of a single A:T base pair to alkylation-induced mutation in a nonexpressed gene, (2) the role of cell proliferation in somatic mutagenesis, and (3) provide a model for a transgenic approach for study of site-specific mutagenesis in vivo in higher eukaryotes.