Analysis on the molecular mechanism of development using insertional mutant mice.

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One insertional mutant showing facial dysplisia was established from transgenic mice carryng human mutant transthyretin genes. This mutant was designated as Nax mouse because nasal and prdmaxillary bones were mainly deformed and this phenotype was transmitted in an autosomal dominant manner. The integration site of transgene was found to be the A5 region of chromosome 13. The cloning of disrupted gene is now under progress.In order to carry out gene trap in embryonic stem (ES) cells, gene trap vector was constructed. This vector contains lacZ gene as a reporter gene, neo-resistant (neo^R) gene as a selection marker gene, and plasmid sequence including replication origin and polycloning site. These vectors were electroplated into ES cells and neo^R clones were selected in the presence of G418. ES cells which show expression of lacZ before or after induction of differentiation were used for isolation of upstream region flanking the insertion site. Upstream DNA sequences ranges from 0.6 to 3 kb. One of these clone termed as Ayu-1 shows a tissue-specific pattern of expression in adult tissues. The cloning of c DNA is now underway.We also improved the method for producing germ-line chimeras. We established LS-10 STO line by introducing neo^R gene and found that these feeder cells are useful for maintaining ES cells without causing differentiation. By the use of transient expression system we obtained culture medium containing high titer of leukemia inhibitory factor. So far, we obtained seven germ-line chimeras by introducing gene trapped ES clones ioto recipient blastocysts.