Establishment of "In situ Knock-out" method

我们的目标是建立一种实验系统，通过该系统，目标分子的功能可以在小鼠从器官发生期到新生期以时间和位置特定的方式被抑制(敲除)。经过初步研究，神经内分泌系统中的一种特定拮抗剂被用作特定抑制目标分子的模型试剂。胚胎操作和试剂导入的技术改进：为了延长试剂导入和稳定掺入试剂的时间，将报告基因整合载体导入小鼠胚胎7天至胚胎10天的羊水中，并对基因整合到皮肤中进行了分析。大约四分之一的操纵胚胎是活着出生的，并显示出基因整合。从而建立了从E7到E10的羊水注射系统和从E11到足月的宫外法直接注射胚胎的系统。通过引入吸收珠来延长拮抗剂作用的试验尚未重现效果，需要进一步修改。从初步研究中选择神经肽Y(NPY)和NPY受体1的特异性拮抗剂(NPY A)进行进一步的比较分析。新生儿期用NPY和NPY-A进行的实验表明，与胶质细胞分化相关的细胞活性如My Elin碱性蛋白(MBP)的mRNA水平、MBP免疫阳性细胞数等存在区域特异性差异，而类似的产前实验尚未给出可重复性的结果。综上所述，我们发明了一种实验系统来抑制从胚胎发育到新生儿期的特定分子功能，包括在组织发生期通过宫外法对特定分子功能的抑制。

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.