Establishement of in vivo Rescue Techniques for Mutant Genes by YAC-Transgenesis

本研究的目的是通过YAC-和bac -转基因建立突变基因的体内拯救技术。近年来，突变基因的定位克隆技术得到了发展。定位克隆包括几个实验步骤：突变基因的连锁分析、物理图谱的构建、表达基因片段（外显子或部分cDNA片段）的筛选、突变体与野生型个体之间多态性的鉴定、突变体候选基因的鉴定。我们尝试开发的技术至少可以应用于位置克隆的两个步骤；1)我们将能够直接识别可能包含导致每种突变的基因的YACs或BACs，从而我们将能够消除大多数时间。这是一个耗费金钱的步骤，构建物理图谱，2)我们将能够在几个候选基因中识别出导致突变的基因。本课题建立了可用于显微注射转基因的YAC和BAC克隆的制备方法。然后，我们通过显微注射将YAC-或BAC-DNA引入原核期小鼠卵中。利用YAC-或bac特异性引物进行PCR检测，我们获得了几只高效的YZC-或bac转基因小鼠，但我们尚未获得任何被YAC克隆拯救的活体小鼠。我们发现，我们分离的大多数YAC克隆显示内部缺失，这可能导致负责突变的基因失活。因此，我们决定使用BAC克隆进行转基因。为此，我们分离了BACs，其基因组区域位于YAC内。我们目前正在进行bac转基因，用于体内抢救。

The aim of this research is to establish in vivo rescue techniques for mutant genes by YAC-, and BAC-transgenesis. Recently, positional cloning of the genes responsible for mutants have been developed. The positional cloning contains several experimental steps : e.g.linkage analysis for the mutant genes, construction of physical maps, screening of expressed gene fragments (exons or partial cDNA fragments), identification of polymorphisms between mutant and wild-type individuals, identification of candidate genes for the mutants. The technique that we have tried to develop can be applied to at least two steps of the positional cloning ; 1) we will be able to directly identiry the YACs or BACs which may contain the genes responsible for each mutation, and thus we will be able to eliminate the most time-.and money-consuming step, the construction of physical maps and 2) we will be able to identify the genes responsible for the mutation among several candidate genes.In this research project, we have established the preparation method of YAC and BAC clones which can be applied to the transgenesis by microinjection. Then, we introduced a YAC-, or a BAC-DNA into mouse eggs with pronucleus-stage by microinjection. Transgenes were detected by PCR using YAC-, or BAC-specific primers We obtained several YZC-, or BAC-transgenic mice with high efficiency However, we have not obtained any mice in vivo rescued by YAC clones. We found that most YAC clones that we isolated showed internal deletions, which might cause inactivation of the genes responsible for the mutants. Therefore, we decided to use BAC clones for the transgenesis. To do so, we isolated BACs, the genomic regions of which are located within the YAC.We are currently doing BAC-transgenesis for in vivo rescue.