| Co-Investigator(Kenkyū-buntansha) |
MORIYASU Matsuko PANAFARM LABORATORIES,INVESTIGATOR, 安全性研究所, 研究員
URANO Toru KUMAMOTO UNIVERSITY SCHOOL OF MEDICINE,ASSOCIATE PROFESSOR, 医学部, 助教授 (90101899)
SUZUKI Misao KUMAMOTO UNIVERSITY SCHOOL OF MEDICINE,ASSOCIATE PROFESSOR, 医学部, 助教授 (60253720)
AIZAWA Shinichi KUMAMOTO UNIVERSITY SCHOOL OF MEDICINE,PROFESSOR, 医学部, 教授 (60073011)
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| Research Abstract |
The efficient production of mice carrying mutations is possible by use of germline mutagenesis with chemical mutagents or X-rays. However, their application is limited, because the vast animal housing resource is required.Moreover, the positional cloning methods are required to identify these mutations. An alternative approach is mutagenesis in embryonic stem (ES) cell. Since 1989, when the first mice were derived with a locus specifically modified by homologous recombination in ES cells, these techniques have been used to mutate approximatery 1% of the total predicted number of mouse loci. However, homologous recombination is laborious and time-consuming. A more attracting approach is random insertional mutagenesis in ES cells using gene trap construct. To carry out the gene trap experiments, we developed a new screening system in which ES cells are differentiated into embryoid bodies (EB) in suspension culture. We found that the patterns of endoderm gene expression during EB development reflect the order found during mouse development in vivo. Thus, we used EB formation to search new genes. We also developed a new site-specific integration system using the Cre-lox recombination system of bacteriophage P1. The lox site is composed of an asymmetric 8 bp spacer flanked by 13 bp inverted repeats. We introduced nucleotide changes in the left 13 bp element (LE mutant lox site) or the right 13 bp element (RE mutant lox site). Recombination between the LE mutant lox site and RE mutant lox site produces a wild-type and a LE+RE mutant site that is poorly recognized by Cre, resulting in stable integration. By applying these new system to the gene trap, we isolated and determined the DNA sequences from 10 trap clones. Four of these are known genes, four are homolougous to ESTs, and two are unknown genes, but have sequence homology to ESTs. Thus, our gene trap system is quite efficient for identifying new genes and producing mutant mice.
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