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Molecular analysis of cystic kidney formed transgenic mouse generated by insertional mutation

插入突变产生的囊性肾形成转基因小鼠的分子分析

基本信息

批准号：
09671183
负责人：
TSUCHIYA Ken
金额：
$ 1.73万
依托单位：
Tokyo Women's Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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-09671183/
关键词：
Polystic kidney Transgenic mouse Ankyrin モノクロナル抗体 NalkATPase Na K ATPase

项目摘要

Vertebrate organisms have a common left-right asymmetry of their visceral organs. However, all unpaired organs of the chest and abdomen, such asd heart, stomach, spleen and liver, develop from the midline in the fetus and localize to their normal positions in the adult. The mirror immage reversal of this asymmetry is called situs inversus. In this study, the inversion of embryonic turning (inv) mutation in a mouse was created by random insertional mutagenesis. The phenotype of the inv mouse is a consistent mirror-image reversal of the left-right polarity (situs inversus) and cystic formation of the kidneys.To analyze the transgenic integration site, the ICRF YAC clones was screened by hybridization with the probe which contains the transgenic integration site. Three YACs were identified and cosmid libraries were constructed from these YACs. Since genomic deletion were indicated, cosmid conting spanning the whole delected region was generated by genomic walking.. In an effort to identif … More y transcript, we used cosmid insert DNA to screen mouse embryo cDNA libraries directly after pre-competition with Cot1 DNA to suppress nonspecific signal. Only one cDNA clone was identified, and then obtained full length cDNA. Northern hybridizations showed that the gene is expressed as early as embroynic day 7, and its size was approximately 5.6 kb. The deduced aminoacid of the gene has revealed ankyrin-like motif in its N-terminal domain.We also analyzed the pathological findings of cyst-formed kidney associated with an inversion of embryonic turning.. We obtained homozygous one day mice after birth and compared them with wild mice. A striking feature of the mutant mouse kidney was the occurrence of various sized tubular cysts. Flattened epithelim with some cells of cuboidal shape, frequent absence of micovilli and dislocation of irregularly shaped mitochondria were observed by electron microscopic examination. A thickened basement membrane was prominent in the cystic tubule. Double staininng with lectin was used to distinguish nephron segments and cyst-formation was shown to occurred mainly in the distal tubule. Both subunits of Na/K ATPase and fodrin were stained at basolateral side, but the staining was faint in large cysts. Cytokeratin was also weakly stained in cells in cystic tubule. In conclusion, the inv mutation mouse consistently replicated multicystformed kidneys. As protein encoded by a candidate gene involved 15 consecutive repeats of ankyrin motif, there may be a possibility that a cytoskeletal abnormality was involved in the mechanism and production of both structural abnormalities, inversion and cyst formation in the kidney. Less

脊椎动物的内脏器官有一个共同的左右不对称性。然而，胸部和腹部的所有不成对的器官，例如心脏、胃、脾和肝脏，都是从胎儿的中线发育出来的，并在成人中定位到其正常位置。这种不对称性的镜像反转称为位置反转。在这项研究中，小鼠胚胎翻转（inv）突变的反转是通过随机插入诱变产生的。 inv小鼠的表型是左右极性（反位）和肾脏囊性形成的一致镜像反转。为了分析转基因整合位点，通过与含有转基因整合位点的探针杂交来筛选ICRF YAC克隆。鉴定了三个 YAC，并从这些 YAC 构建了粘粒文库。由于表明基因组缺失，通过基因组行走生成了跨越整个缺失区域的粘粒序列。为了鉴定 y 转录本，我们在与 Cot1 DNA 预竞争后直接使用粘粒插入 DNA 筛选小鼠胚胎 cDNA 文库，以抑制非特异性信号。仅鉴定出1个cDNA克隆，然后获得全长cDNA。 Northern杂交表明该基因早在胚胎第7天就已表达，其大小约为5.6 kb。推导的该基因的氨基酸在其N末端结构域中揭示了锚蛋白样基序。我们还分析了与胚胎转向反转相关的囊肿形成肾的病理结果。我们获得出生后一天的纯合小鼠，并将其与野生小鼠进行比较。突变小鼠肾脏的一个显着特征是出现各种大小的管状囊肿。电镜观察可见上皮扁平，部分细胞呈立方体形状，微绒毛经常缺失，线粒体错位，形状不规则。囊性小管中有明显增厚的基底膜。使用凝集素双重染色来区分肾单位节段，并且显示囊肿形成主要发生在远端肾小管中。 Na/K ATPase和fodrin的两个亚基在基底外侧均被染色，但在大囊肿中染色较弱。囊性小管细胞中的细胞角蛋白也被弱染色。总之，inv 突变小鼠持续复制多囊形成的肾脏。由于候选基因编码的蛋白质涉及锚蛋白基序的15个连续重复，因此细胞骨架异常可能参与肾脏结构异常、倒位和囊肿形成的机制和产生。较少的