Positional cloning of mouse morphological mutants

小鼠形态突变体的定位克隆

• 批准号: 09440257
• 负责人: SHIROISHI Toshihiko
• 金额: $ 8.7万
• 依托单位: National Institute of Genetics
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09440257/
• 关键词: Tail-short (Ts); morphological anomalies; homeotic transformation; positional cloning; cDNA selection; ribosomal protein; Rpl38; transgenesis; リボソーム蛋白; Rpl38遺伝子; トランスジェネシス; Rim4変異、Hx変異; マイクロサテライト; BACクローン; shh遺伝子; 形態形成; マウス; 突然変異; 連鎖解析 /

A mouse mutation Tail-short (Ts) exhibits shortened kinky tail and numerous skeletal abnormalities including homeotic anteroposterior patterning problem along the axial skeleton. Ts gene was mapped to the teromeric region of the chromosome 11. To elucidate the function of the Ts gene in mouse embryogenesis, we intended to clone the gene by means of positional cloning. First, we employed a fine mapping of this gene based on a large scale intersubspecific backcross between the mutant stock TsJ/Le-Ts/ + and Japanese wild mouse-derived MSM strains. Ts gene was mapped to a 0.16cM region between two microsatellite markers, D1IMit128 and DlIMit256. We screened mouse YAC and BAC libraries with microsatellite markers tightly linked to the Ts locus and have obtained YAC and BAC clones. Further chromosome walking with the isolated clones allowed us to construct a complete BAC contig covering the Ts causative gene and the critical region of the Ts was narrowed between two new STSs, DI IRin56 and D … More lINigI7. This contig consists of 3 BAC clones, spanning a 250kb DNA fragment. We have isolated several cDNA clones as Ts candidate gene from the critical region by directed cDNA selection using the corresponding BAC. We have obtained several clones derived from mouse embryonic cDNA library. Among these candidate genes, the expression level of Rp138 gene, which encodes one of ribosomal protein subunits, was reduced in Ts mutant mouse. Analysis of the genomic structure of the Ts mutant revealed that it had an 18kb long deletion containing Rpl38. Other two mutations, Rabo torcido (Rbt) and Tail-short shionogi (Tss), were reported to have phenotype similar to Ts and to be mapped to the same region In the chromosome 11. We found that Rbt and Tss have a frame shift mutation and an insertional mutation, respectively. In order to confirm that Rpl38 is the causative gene for Ts, we tried to rescue the Ts mutant lethal phenotype by transgenesis with the wild-type gene of Rpl38. Over expression both of the BAC containing Rpl38 and the Rpl38 cDNA restored the viability of the Ts mutant in a certain genetic background and completely rescued the morphological phenotype. Thus, it appeared that the lethality and the morphological anomalies of Ts mutants, including their homeotic transformation, are caused by the Rpl38 mutation. Less

小鼠尾短突变 (Ts) 表现出缩短的扭尾和许多骨骼异常，包括沿中轴骨骼的同源异型前后图案问题。 Ts基因定位于11号染色体的三聚体区域。为了阐明Ts基因在小鼠胚胎发生中的功能，我们打算通过定位克隆的方式克隆该基因。首先，我们基于突变体 TsJ/Le-Ts/ + 和日本野生小鼠来源的 MSM 品系之间的大规模亚种间回交，对该基因进行了精细作图。 Ts 基因被定位到两个微卫星标记 D1IMit128 和 DlIMit256 之间的 0.16cM 区域。我们用与Ts位点紧密连锁的微卫星标记筛选小鼠YAC和BAC文库，并获得了YAC和BAC克隆。使用分离的克隆进行进一步的染色体行走使我们能够构建覆盖 Ts 致病基因的完整 BAC 重叠群，并且 Ts 的关键区域在两个新的 STS（DI IRin56 和 D … 更多 lINigI7）之间缩小。该重叠群由 3 个 BAC 克隆组成，跨越 250kb DNA 片段。我们通过使用相应的 BAC 进行定向 cDNA 选择，从关键区域分离了几个 cDNA 克隆作为 Ts 候选基因。我们已经从小鼠胚胎 cDNA 文库中获得了几个克隆。在这些候选基因中，编码核糖体蛋白亚基之一的Rp138基因的表达水平在Ts突变小鼠中降低。对Ts突变体的基因组结构分析表明，它有一个包含Rpl38的18kb长的缺失。据报道，另外两个突变 Rabo torcido (Rbt) 和 Tail-short shionogi (Tss) 具有与 Ts 相似的表型，并且定位到 11 号染色体的同一区域。我们发现 Rbt 和 Tss 分别具有移码突变和插入突变。为了证实Rpl38是Ts的致病基因，我们尝试通过转基因Rpl38的野生型基因来挽救Ts突变致死表型。含有Rpl38的BAC和Rpl38 cDNA的过表达都恢复了Ts突变体在一定遗传背景下的活力，并完全挽救了形态表型。因此，Ts突变体的致死率和形态异常，包括它们的同源异型转化，似乎是由Rpl38突变引起的。较少的

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