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

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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.