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DEVELOPMENTAL GENETIC ANALYSIS OF MAMMALIAN GENOME FUNCTIONS

哺乳动物基因组功能的发育遗传分析

基本信息

批准号：
05671885
负责人：
ABE Kuniya
金额：
$ 1.34万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671885/
关键词：
DEVELOPMENT YAC CLONING TRANSGENIC MOUSE 4T t COMPLEX 発生分化 H-2 tコンプレックス YAC クローニングトランスジェニックマウスゲノムクローニング H-2K

项目摘要

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

定位克隆方法已成功分离出各种突变基因，包括负责肿瘤发生的基因。然而，尽管基因作图和基因组克隆技术取得了如此成功并取得了最新进展，突变基因的鉴定和分离仍然是一项艰巨的任务。如果没有发现总的基因组重排，例如易位或缺失，则很难在大的基因组片段中查明负责的基因。因此，对克隆基因组 DNA 进行功能表征的方法的需求越来越大。在这项研究中，已经建立了一种能够从大型克隆基因组 DNA 中快速恢复转录序列的方法。此外，为了对大基因组 DNA 进行功能表征，正在开发一种生成携带 YAC DNA 的转基因小鼠的技术。小鼠的 T/t 复合体是 17 号染色体近半部分的一个大遗传区域，携带许多影响胚胎发生和生殖细胞功能的基因座。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，我们对诸如 st^<omega5> 等突变特别感兴趣，这是一种隐性植入后致死突变，显示胚胎外胚层形成缺陷； qk（颤抖），一种神经突变体。我们已将 t^<omega5> 映射到小鼠 MHC 区域 H-2 复合体内。 t^<omega5>被发现与H-2K基因非常接近，遗传距离可能小于0.1cM。我们将K基因周围跨越-200kb的基因组区域克隆到粘粒重叠群中，并在克隆区域内搜索基因。现在，我们通过分离几个覆盖从 K 基因到 CryA1 标记 -800kb 的 YAC 克隆来扩展克隆区域。遗传分析表明突变存在于克隆的DNA中，但迄今为止该区域尚未发现结构改变。通过使用我们的“溶液筛选”方法，在这个克隆的基因组区域中发现了 22 个新基因。此外，为了找到负责的基因，还应用了将 YAC DNA 引入种系的技术。我们首先建立了不断裂的大DNA片段的分离和纯化方案，从而将从t^<omega5>区域分离的650kb YAC注射到小鼠受精卵中。将 320 个注射后的卵转移到假孕寄养家庭后，39 只幼崽诞生了。其中 3 只小鼠至少携带部分 YAC 载体序列，表明转基因整合。现在正在对这些假定的转基因小鼠的基因组 DNA 进行详细表征。较少的