A NEW TRANSGENIC MOUSE MUTAGENESIS TEST SYSTEM USING SPI(-) AND 6-THIOGUANINE SELECTIONS

A new transgenic mouse mutagenesis test system has been developed For the efficient detection of point mutations and deletion mutations in v ivo. The mice carry lambda EG10 DNA as a transgene. When the rescued p hages are infected into Escherichia coli YG6020-expressing Cre recombi nase, the phage DNA is converted into plasmid pYG142 carrying the chlo ramphenicol-resistance gene and the gpi gene of E. coli. The gpt mutan ts can be positively detected as colonies arising on plates containing chloramphenicol and 6-throguanine. The EG10 DNA carries a chi site al ong with the red and gam genes so that the wild-type phages display Sp i(+) (sensitive to P2 interference) phenotype. Mutant phages lacking b oth red and gam genes can be positively detected as plaques that grow in P2 lysogens of E. coli. These mutant phages are called lambda Spi(- ). The spontaneous gpi mutation frequencies of five independent transg enic lines were 1.7 to 3.3 x 10(-5) in bone marrow. When the mice were treated with ethylnitrosourea (single i.p. treatments with 150 mg/kg body weight; killed 7 days after the treatments), mutation frequencies were increased four- to sevenfold over the background in bone marrow. The average rescue efficiencies were more than 200,000 chloramphenico l-resistant colonies per 7.5 mu g bone marrow DNA per packaging reacti on. In contrast to gpi mutation frequencies, spontaneous Spi(-) mutati on Frequencies were 1.4 x 10(-6) and 1.1 x 10(-6) in bone marrow and s perm, respectively. No spontaneous Spi(-) mutants have been detected s o far in spleen, although 930,000 phages rescued from untreated mice w ere screened. In gamma-ray-treated animals, however, induction of Spi( -) mutations was clearly observed in spleen, at frequencies of 1.4 x 1 0(-5) (5 Gy), 1.2 x 10(-5) (10 GY), and 2.0 x 10(-5) (50 Gy). These re sults suggest that the new transgenic mouse ''gpt delta'' could be use ful for the efficient detection of point mutations and deletion mutati ons in vivo. (C) 1996 Wiley-Liss, Inc.