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

脊椎动物的内脏器官通常是左右不对称的。而胸腹各不配对的器官，如心、胃、脾、肝等，在胎儿时从中线发育，在成人时定位到正常位置。这种不对称的镜像反转称为位置反转。在本研究中，通过随机插入突变在小鼠中产生了胚胎转向反转（inv）突变。inv小鼠的表型是左-右极性（倒置位置）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，用含有转基因整合位点的探针对ICRF YAC克隆进行杂交筛选。鉴定了3个YACs并构建了cosmid文库。由于基因组缺失被指出，跨越整个缺失区域的基因组序列是通过基因组行走产生的。为了鉴定更多的转录本，我们使用cosmid插入DNA在与Cot1 DNA竞争后直接筛选小鼠胚胎cDNA文库，以抑制非特异性信号。只鉴定了一个cDNA克隆，就得到了全长cDNA。Northern杂交表明，该基因早在第7天就开始表达，其大小约为5.6 kb。该基因的氨基酸在其n端结构域显示出锚蛋白样基序。我们还分析了囊肿形成的肾脏与胚胎转向反转相关的病理结果。我们获得出生后1天的纯合子小鼠，并与野生小鼠进行比较。突变小鼠肾脏的一个显著特征是出现各种大小的管状囊肿。电镜观察上皮扁平，部分细胞呈立方状，微绒毛缺失，不规则线粒体脱位。囊小管基底膜增厚。用凝集素双染色区分肾元节段，显示囊肿主要发生在远端小管。基底外侧均可见Na/K atp酶亚基和fodrin亚基染色，但在大囊肿中染色较弱。囊小管细胞角蛋白染色较弱。总之，突变小鼠一致地复制了多系统肾脏。由于候选基因编码的蛋白涉及锚蛋白基序的15个连续重复，因此可能存在细胞骨架异常参与肾脏结构异常、倒置和囊肿形成的机制和产生。少

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