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

脊椎动物的内脏器官有一个共同的左右不对称。然而，胸腹部的所有未配对器官，如心、胃、脾和肝，在胎儿时都是从中线发育，到成人时则位于正常位置。这种不对称性的镜像反转称为反转位。本研究采用随机插入突变法，在小鼠中产生了胚胎翻转倒位（inv）突变。inv小鼠的表型是一致的左右极性的镜像反转（反位）和肾脏的囊性形成。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筛选ICRF YAC克隆。鉴定了三个YAC，并从这些YAC构建粘粒文库。由于指示基因组缺失，因此通过基因组步移产生跨越整个缺失区域的粘粒。为了确定 ...更多信息 y转录本的cDNA文库中，我们用粘粒插入DNA与Cot 1 DNA预竞争以抑制非特异性信号后直接筛选小鼠胚胎cDNA文库。仅鉴定了一个cDNA克隆，然后获得全长cDNA。北方杂交结果表明，该基因在胚胎发育的第7天就开始表达，大小约为5.6kb。该基因的N-末端氨基酸序列具有类似锚定蛋白的结构域，并分析了与胚胎翻转倒置相关的囊肿形成肾的病理学表现。我们获得了出生后一天的纯合子小鼠，并将其与野生小鼠进行了比较。突变小鼠肾脏的一个显著特征是发生各种大小的管状囊肿。电镜下见上皮扁平，部分细胞呈立方状，常无微绒毛，线粒体排列不规则。囊小管基底膜增厚明显。用凝集素双染区分肾单位节段，囊肿形成主要发生在远端小管。Na/K ATP酶和胞衬蛋白在基底外侧均有表达，但在大囊肿中染色较弱。细胞角蛋白在囊小管内也呈弱染色。总之，inv突变小鼠一致地复制了多囊肾。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，因此可能存在细胞骨架异常参与肾脏结构异常、倒位和囊肿形成的机制和产生的可能性。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