DEVELOPMENTAL GENETIC ANALYSIS OF MAMMALIAN GENOME FUNCTIONS

定位克隆方法已成功地分离了多种突变基因，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，但鉴定和分离突变基因仍然是一项艰巨的任务。在没有发现总体基因组重排（例如易位或缺失）的情况下，很难在一个大的基因组片段中确定一个负责任的基因。因此，对克隆基因组DNA功能表征方法的需求日益增加。在这项研究中，一种方法，允许快速恢复转录序列从大克隆基因组DNA已经建立。此外，为了对大基因组DNA进行功能表征，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号染色体近一半的一个大的遗传区域，它携带了许多影响胚胎发生和生殖细胞功能的位点。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。在这些突变中，我们特别感兴趣的是ast^<omega5>，这是一种隐性的植入后致死突变，显示出胚胎外胚层形成的缺陷；Qk（颤抖），神经突变体。我们在小鼠MHC区域H-2复合体中绘制了t^<omega5>。发现t^<omega5>与H-2K基因非常接近，遗传距离可能小于0.1cM。我们克隆了K基因周围-200kb的基因组区域，并对克隆区域内的基因进行了搜索。现在，我们通过从k基因向CryA1标记分离几个覆盖-800kb的YAC克隆来扩展克隆区域。基因分析表明，突变存在于克隆DNA中，而到目前为止在该区域未发现结构变化。利用“溶液筛选”方法在该克隆基因组区发现了22个新基因。此外，为了找到负责基因，还采用了将YAC DNA引入种系的技术。我们首先建立了无断裂大片段DNA的分离纯化方案，从而从t^<omega5>区分离出650 kb的YAC，注射到小鼠受精卵中。在将320个注射的卵子移植给假怀孕的养犬后，39只幼崽出生了。其中3只小鼠至少携带部分YAC载体序列，提示存在转基因整合。这些假定的转基因小鼠的基因组DNA现在正在被详细地表征。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