Nucleic Acids Research, 1990, Vol. 18, No. 10 3007
© 1990
MOLECULAR BIOLOGY |
Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice
Stratagene, 11099 N. Torrey Pines Road, La Jolla, CA 92037, USA
*To whom correspondence should be addressed
Received November 22, 1989. Accepted March 13, 1990.
Transgenic mice suitable for the in vivo assay of uspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a /S-galactosidase (/3-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations In the target gene are detected by a change In lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100/ig of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytldine-treated cells increased 50-fold over nontreated controls Indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coll plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/µxg of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1x10–5 in two separate lineages. Following treatment with the mutagen N-ethyl-rV-nitrosourea (EtNU), a dose dependent increase In the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed In mouse testes DNA
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