DEVELOPMENTAL GENETIC ANALYSIS OF MAMMALIAN GENOME FUNCTIONS

定位克隆方法已成功地分离出各种突变基因，包括与肿瘤发生有关的基因。然而，尽管在基因定位和基因组克隆技术方面取得了这些成功和最新进展，但突变基因的鉴定和分离仍然是一项艰巨的任务。在没有发现总的基因组重排（例如易位或缺失）的情况下，很难在大的基因组区段内精确定位负责基因。因此，有一种方法，以克隆基因组DNA的功能特性的日益增长的需求。在这项研究中，已经建立了一种方法，允许从大克隆基因组DNA的转录序列的快速恢复。此外，为了在功能上表征大基因组DNA，正在开发一种产生携带YAC DNA的转基因小鼠的技术。小鼠的T/t复合体是位于17号染色体近端的一个大的遗传区域，其携带许多影响胚胎发生的基因座， ...更多信息 生殖细胞功能我们一直在分析这个区域，试图最终分离出导致这些突变的基因。其中，我们特别感兴趣的突变，如ast^<omega5>，一种隐性的植入后致死突变，显示胚胎外胚层形成缺陷; qk（震颤），一种神经突变。我们已经<omega5>在小鼠MHC区域H-2复合体中定位了t^。我们<omega5>克隆了K基因周围约200 kb的基因组区域，并在克隆区域内进行了基因搜索。现在，我们通过分离几个YAC克隆来扩展克隆区域，所述YAC克隆覆盖从K基因到CryA 1标记的~ 800 kb。遗传分析表明，突变位于克隆的DNA中，而迄今为止在该区域没有发现结构改变。利用我们的“溶液筛选”方法，在该克隆基因组区域发现了22个新基因。此外，为了找到一个负责的基因，一种技术，将YAC DNA导入生殖系已被应用。我们首先建立了一种不断裂的大片段DNA的分离纯化方法，并将分离的650 kb YAC<omega5>注射到小鼠受精卵中。在将320个注射的卵子转移到假孕寄养后，出生了39只幼崽。其中3只小鼠携带至少一部分YAC载体序列，表明转基因整合。目前正在对这些假定的转基因小鼠的基因组DNA进行详细的鉴定。少

Positional cloning approach has been successful in isolation of mutated genes of various kinds, including genes responsible for tumorigenesis. However, despite such successes and recent progress in gene mapping and genomic cloning technologies, the identification and isolation of mutated genes still remains formidable task. In cases no gross genomic rearrangements e.g.translocation or deletion are found, it is very difficult to pinpoint a responsible gene within a large genomic segment. Yhus, there is an increasing demand for a method to functionally characterize the cloned genomic DNA.In this study, a method that allows rapid recovery of transcribed sequences from large cloned genomic DNA has been established. Also, in order to functionally characterize large genomic DNA,a technique to generate transgenic mice carrying YAC DNA is being developed.The T/t complex of mouse is a large genetic region on proximal half of chromosome 17 that carry a number of loci affecting embryogenesis and … More germ cell functions. We have been analyzing this region, attempting to ultimately isolate genes responsible for these mutations. Among these, we have special interests in mutations such ast^<omega5>, a recessive postimplantation lethal, showing defects in embryonic ectoderm formation ; qk (quaking), a neurological mutant. We have mapped t^<omega5> within mouse MHC region, H-2 complex. t^<omega5> was found to be very close to the H-2K gene with a possible genetic distance less than 0.1cM.We have cloned genomic region spanning -200kb around the K gene into cosmid contigs and have searched genes within the cloned region. Now, we extended the cloned area by isolating several YAC clones that cover -800kb from the K-gene toward CryA1 marker. Genetic analysis suggests that the mutation lies within the cloned DNA,while no structural alterations are found so far in this region. Twenty two novel genes have been found in this cloned genomic area by using our' screening in solution' method. Also, to find a responsible gene, a technique to introduce YAC DNA into germ line has been applied. We first established a protocol for isolation and purification of large DNA fragment without breakage, and thus isolated 650 kb YAC from t^<omega5> region was injected into mouse fertilized eggs. After transferring 320 injected eggs to pseudopregnant foster, 39 pups were born. Of which 3 mice carry at least a part of the YAC vector sequence, suggesting transgene integration. Genomic DNA from these putative transgenic mice are now being characterized in detail. Less