Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice.
Transgenic mice with a lambda shuttle vector containing a lacI target gene were generated for use as a short-term, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal). Phage with mutations in the lacI gene form blue plaques, whereas phage with a nonmutated lacI form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10(-5) to 1.7 x 10(-5), depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the mice with N-ethyl-N-nitrosourea (EtNU), benzo[a]pyrene (B[a]P), or cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the lacI gene, when replicated in Escherichia coli, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the lacI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance of mutations (81%) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.
Kohler
S W
SW
Stratagene, La Jolla, CA 92037.
Provost
G S
GS
Fieck
A
A
Kretz
P L
PL
Bullock
W O
WO
Sorge
J A
JA
Putman
D L
DL
Short
J M
JM
D000818:Animals; D010582:Bacteriophage lambda; D001564:Benzo(a)pyrene / Q000494:pharmacology; D003520:Cyclophosphamide / Q000494:pharmacology; D005038:Ethylnitrosourea / Q000494:pharmacology; D005809:Genes, Regulator; D005822:Genetic Vectors; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D016296:Mutagenesis; D009152:Mutagenicity Tests / Q000379:methods*; D012097:Repressor Proteins / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D012097:Repressor Proteins; D001564:Benzo(a)pyrene; D003520:Cyclophosphamide; D001616:beta-Galactosidase; D005038:Ethylnitrosourea