International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)

Table of Contents * Complex Genetics * Developmental Genetics * Gene Annotation * Gene Discovery * Genome Sequencing * Functional Genomics * Mutagenesis * Presentations * Verne Chapman Memorial Lecture

Dr Jun-ichi Asakawa
Radiation Effects Research Foundation
5-2 Hijiyama-Park, Minami-ku
732-0815 Hiroshima Japan

Co-Author:  Itoh M 
Institution:   Department of Genetics, Radiation Effects Research Foundation

We have developed two-dimensional electrophoresis (2-DE) technique of DNA to study spontaneous and radiation-induced mutations in mouse germ cells. The original method utilized NotI enzyme to end-label CpG rich DNA fragments. To detect mutations at genomic regions other than CpG islands, we developed a method to use AflII (C/TTAAG) as the landmark enzyme in place of NotI. In the new method, DNA was first digested with AflII/HaeIII (GG/CC), 3-8kb fragments were isolated and radio-labeled at the AflII sites. After the first dimension electrophoresis, the DNA fragments were further digested in situ with HinfI and separated by the second dimension electrophoresis. The 2-DE patterns revealed that, in the F1 animals from the cross of JF1 (Japanese Fancy Mouse 1, an inbred strain originated from the Japanese wild mouse, M. m. molossinus) x B6C3F1, numerous spots derived from B6C3F1 mice were heterozygous. Namely, among 1415 independent spots from B6C3F1 mice, 783 were homozygous while 632 were heterozygous (among the latter, 318 were derived from B6 and 314 were from C3H). In F1 of another cross, JF1 x BALB/c, only about 15% were homozygous among approximately 2000 spots. The results indicate that AflII/HaeIII/HinfI fragments visualized on a single DNA 2-DE gel are highly polymorphic among mouse strains and the frequency of inter-strain variation seemed to be comparable to that of microsatellite markers.