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

小鼠突变尾短（Ts）表现出缩短的弯曲尾巴和许多骨骼异常，包括沿着中轴骨骼的同源异型前后图案问题（homeotic anteroorbital patterning problem）沿着骨骼。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法克隆该基因。首先，我们采用了一个精细的映射，这个基因的基础上大规模的亚种间回交突变股票TsJ/Le-Ts/ +和日本野生小鼠来源的MSM菌株。Ts基因定位在D1 IMit 128和D1 IMit 256两个微卫星标记之间的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，获得了YAC和BAC克隆。用分离的克隆进一步进行染色体步移，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，DIIRin 56和D ...更多信息 linigI 7.该重叠群由3个BAC克隆组成，跨越250 kb的DNA片段。我们利用相应的BAC进行cDNA定向选择，从关键区域中分离出了几个作为Ts候选基因的cDNA克隆。我们从小鼠胚胎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Rp 138基因在Ts突变小鼠中表达水平降低。对Ts突变体的基因组结构的分析揭示，它具有包含Rpl 38的18 kb长的缺失。其他两个突变，Rabo torcido（Rbt）和Tail-short shionogi（Tss），据报道具有与Ts相似的表型，并定位于11号染色体的相同区域。我们发现Rbt和Tss分别有一个移码突变和一个插入突变。为了证实Rpl 38是Ts的致病基因，我们试图通过用Rpl 38的野生型基因转基因来拯救Ts突变体致死表型。过表达含有Rp 138的BAC和Rp 138 cDNA两者恢复了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，看来Ts突变体的致死性和形态异常，包括它们的同源异型转化，是由Rp 138突变引起的。少

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