| Research Abstract |
Using the recombination system Cre-loxP derived from bacteriophage P1, we developed the gene insertion system. This system comprises of two mutant lox, lox71 and lox66. We constructed a new trap vector that carry this lox71. The method using this trap vector was termed as the exchangeable gene trap, because we can insert any gene of interest after gene trapping. We alaso demonstrated that the efficiency of gene integration was improved by combination of mutant lox71/66 and mutant lox511 that carry mutation in the spacer region. ES cells can be induced to differentiate to form embryoid bodies by removing from feder cells and maintaining in suspension culture. We used this embryoid body formation as a screening system to trap genes involved in cell proliferation and early stages of development. We established 24 trap lines. Among these half of them showed lethilty in development and growth. We isolated the genes trapped by trap vector and tne nucleotide sequences were determined. These s
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eqences were compared with DNA database using balst program. These include several classes of proteins involved in transcription, cell growth, cell division, singal transduction, translation and transportaiton. Among trap lines established, we analyzed two lines in which CBP (creb binding protein) and c-crk genes are disrupted. A heterozygous mouse for CBP mutation is a mouse model for human disease, Rubinstein-Taybi syndrome, and shows most of phenotypes found in human patient. A homozygous mouse is embryonic lethal due to failures of hematopoiesis and vasculogenesis. A homozygous mouse for c-crk mutation did not show any phenotype. However, they died within 24 hours after birth by backcrossing to C57BL/6 suggesting the presence of modifier gene. A method for microinjecting bacterial artificial chromosome has been established and 34 strains of mice that carry bacterial artificial chromosome were generated. Among these, 31 cases are found to have complete bacterial artificial chromosome without any deletion. BACs containing T gene and qkI gene were shown to rescue T mutant and qk mutant, respectively. Less
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