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

YAC转基因体内突变基因拯救技术的建立

基本信息

批准号：
07458231
负责人：
YONEKAWA Hiromichi
金额：
$ 4.99万
依托单位：
The Tokyo Metropolitan Institute of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

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.

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。最近，已经开发了负责突变体的基因的定位克隆。定位克隆包括几个实验步骤：例如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，突变体和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可以应用于定位克隆的至少两个步骤; 1）我们将能够直接鉴定可能含有导致每个突变的基因的YAC或BAC，因此我们将能够消除最耗时和最费钱的步骤，物理图谱的构建和2）我们将能够在几个候选基因中识别出负责突变的基因。在这个研究项目中，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，我们用显微注射法将YAC-或BAC-DNA导入小鼠原核期的卵细胞中。我们获得了几只高效率的YZC或BAC转基因小鼠，然而，我们还没有获得任何体内被YAC克隆拯救的小鼠。我们发现，我们分离的大多数YAC克隆显示内部缺失，这可能会导致负责突变体的基因失活。因此，我们决定使用BAC克隆进行转基因。为此，我们分离了BAC，其基因组区域位于YAC内。我们目前正在进行BAC转基因用于体内拯救。