International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)


H5. A novel Strategy for Mutagenesis in the Mouse : Heavy Ion Beams and Fetal Phenotype Screening -------II. Production and Screening

Atsushi Yoshiki, Chikako Yoshida-Noro, Hiroki Nagase#, Noriko Hiraiwa, Aya Kogiso, Takanobu Masuda, Moriaki Kusakabe
Experimental Animal Research Division and #Molecular Oncology Laboratory, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
E-mail: kusakabe@rtc.riken.go.jp
Tel:+81-(0)298-36-5264; Fax:+81-(0)298-36-9010

In this paper we focused on the production and screening of recessive fetal phenotypes by heavy ion beams. Males of C57BL/6J (B6) strain mice were anesthetized, held on a plastic plate and irradiated with C, N, and Ne ion beams at dosages of 3 and 5 Gy. The exposure to the heavy ion beams was restricted to the inguinal region of the body. Six weeks later successful irradiation of the testes was indicated by coat color changes in the ventral and dorsal skin. Histological study on testis tissues indicated that germ cells in seminiferous epithelium were significantly affected with C, N, and Ne ion beams at 3-5 Gy. Following the sterile periods, some male mice could restart reproduction. These irradiated G0 males were crossed with nonirradiated females to create G1 founders. Subsequently, the G1 founders were crossed with females to produce G2 females, half of which were heterozygous for the newly induced mutations inherited from the G1 founders. Then, G2 females were backcrossed with their fathers, and sacrificed on days 10-13 of gestation for the screening of morphological abnormalities in G3 embryos. To date, we have examined 28 G1 males, and determined that 25 were fertile. With the 25 fertile G1 males, 226 G2 females became pregnant, and had 1689 morphologically normal embryos (88.2%), 107 morphologically abnormal ones (5.6%), and 119 absorption sites (6.2%). These morphological phenotypes were expected to be multigenic and includes developmental retardation at E8-9, yolk-sac-like structure without a proper of embryo, defect in neural tube closure, and craniofacial malformation. Morphologically abnormal embryos were subjected to constuct micro-tissue subtraction cDNA library to extract a characteristic expression profile relating to each unique morphological phenotypes. We will present our detailed technical strategy for production and screening of mutant by heavy ion beam and discuss its unique status as useful resource for the functional genomics.


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