|Budget Amount *help
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1996: ¥2,100,000 (Direct Cost: ¥2,100,000)
Insertional mutagenesis is commonly used to generate mutants in a systemic manner. Establishment of embryonic stem cells and development of gene trap vectors have made it possible to apply insertional mutagenesis for generation of mutant mice. Therefore, systematic mutagenesis of mice using insertional mutagenesis is currently in a scope of geneticists including us. Although several mutants have been successfully established with a retroviral gene-trap vector in our lab, we also encountered several ploblems to initiate a large scale mutagenesis using this vector. ln this study, we constructed two novel vectors and initiated a small scale mutagenesis using these vectors as a feasibility study for a large scale insertional mutagenesis of mice. We have been using ROSA-BETAgeo, a retrovirus vector containing a fusion gene of LacZ and neo, with splice acceptor sequences at its 5' end. First, we constructed a novelvector, ROSA-BETAgeo-loxP, by inserting a loxP sequence into 3'LTR of ROSA-BETAgeo. When provirus DNA integrated into a gene on chromosomal DNA of ES cells, a gene trap machinery, introducing a mutation, was flanked by a pair of loxP sites and, therefore, it was easily deleted from chromosomal DNA by expressing Ore recombinase leaving a single loxP site. Therefore, it is possible to generate reversible mutants with this system. We also generated ROSA-IRES-BETAgeo, another novel vector containing internal ribosome entry sequence in between splice acceptor sequence and BETAgeo gene. Using this novel vector, we isolated mutants carrying gene trap vectors inserted into introns at 3' side of target genes. Therefore, we could expect to generate hypomorphic mutants. Using these novel vectors generated in this study, we could establish more than 20 lines of mutant mice. Four of them were shown to suffer mutations of known genes and 2 lines showed embryonic lethality, suggesting efficient production of mutants in our system.