International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

H13 Mouse Mutagenesis by Heavy Ion Beams: Detection of Mutant Mice by Random Amplified Polymorphic DNA (RAPD)

Teruyo Tsukada, Mio Furutsu, Aya Kogiso, Hirotake Yamaguchi, Yukiko Kitasaka, Masayo Kojima, Noriko Hiraiwa, Atsushi Yoshiki and Moriaki Kusakabe. Division of Experimental Animal Research, The Center for Biogenic Resources, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

Mouse mutants are a quite useful animal model to study the gene functions and pathogenesis of human diseases. However, we do not have enough kinds of mutants with phenotypes that reveal functions of all the genes. For this reason, we need more effort to create and find new phenotypes in mouse mutagenesis. We would like to report an application of heavy ion beams for mouse mutagenesis. However, to analyze mutations by observing phenotypes is very hard to classify in heterozygous state, because many mutations do not appear with any abnormal phenotypes in heterozygous state. A single 10-base random oligonucleotides of arbitrary sequence could be used to prime the amplification of genomic sequences in a reproducible and polymorphic fashion, and this protocol is called random amplified polymorphic DNA (RAPD). A particular DNA fragment that is generated for one individual but not for another represents a DNA polymorphism and can be used as a genetic marker. We think this method is also useful for detection of genomic mutations. Sperm were collected from the caudal epididymis of C57BL/6 mice, and were irradiated by heavy ion beams (40Ar at 95 MeV/u, 56Fe at 90 MeV/u, and 20Ne, 14N and 12C at 135 MeV/u) at RIKEN Ring Cyclotron Facility. Mice were created by sperm head injection. Developed eggs were transferred into oviducts of pseudopregnant mothers to produce G1 mice. We have also tried to induce mutations in male premeiotic germ cells. Testes of B6 male mice were also irradiated by heavy ion beams. These irradiated males were used for mating with non-treated females to produce G1 mice. We examined both of these G1 mice to detect genomic mutations by RAPD analysis. We have seen different banding patterns in amplification products, such as addition, missing, or shifting of bands. Furthermore, we selected heterozygous mutants indicated by banding patterns as potential breeders to produce homozygous mutants. We would like to report the phenotypes of our mutants by screening genotypes with RAPD method.

 


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