Establishment of "In situ Knock-out" method

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We aimed to establish an experimental system by which functions of target molecules can be inhibited (knocked-out) in a time-and position-specific manner in mice from organogenetic to neonatal periods. After pilot studies, a specific antagonist in the neuroendocrine system was employ ed as a model agent for specific inhibition of the target molecule.1. Technical improvement of embryo manipulation and agent introduction.To extend the period of agent introduction and stable incorporation of the introduced agent, a reporter gene integrated vector was introduced into the amniotic fluid of from embryonic day (E) 7 to E10 mouse embryos, and the gene integration into the skin was analyzed. Approximately one fourth of the manipulated embryos were born live, and showed the gene integration. Thus, the injection system into intra-amniotic fluid from E7 to E10, and directly to embryos by exo utero method from E11 to term has been established. The trial to extend the effect of antagonists by introducing absorptive beads has not yet reproducibly worked, and needs further modifications.2. Introduction of a specific inhibitor into embryos and analysis of the effect.Neuropeptide Y (NPY) and a specific antagonist for NPY receptor 1 (NPY A) were selected from pilot studies and used for further comparative analyses. Neonatal period experiments with NPY and NPY-A showed region-specific differences in glia cell differentiation-related cellular activities such as mRNA level of my elin basic protein (MBP), number of MBP immuno-positive cells, whereas similar experiments in prenatal periods have not yet given reproducible results.In summary, an experimental system was invented to inhibit specific molecular function from oranogenetic to neonatal periods, including the time-and site-specific inhibition by exo utero method during the histogenetic period.