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

脊椎动物的内脏器官有常见的左右不对称。然而，胸部和腹部的所有不成对的器官，如心脏、胃、脾和肝脏，都是从胎儿的中线发育起来的，并在成人时定位到正常的位置。这种不对称性的镜像反转称为逆位。在本研究中，通过随机插入突变产生了小鼠胚胎翻转(Inv)突变的倒置。INV小鼠的表型是肾脏左右两极(反位)和囊状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杂交筛选了ICRF YAC克隆。鉴定了3个YAC，并用这些YAC构建了粘粒文库。由于基因组被提示缺失，整个缺失区域的粘粒竞争是通过基因组行走产生的。为了确认…更多的y转录本，我们用粘粒插入DNA直接筛选小鼠胚胎cDNA文库，与Cot1 DNA预竞争以抑制非特异性信号。只鉴定了一个克隆，并获得了全长的cDNA.Northern杂交结果表明，该基因早在胚胎发育第7天就开始表达，大小约为5.6kb。该基因推导的氨基酸序列在其N-末端结构域上显示了类似锚蛋白的基序。我们还分析了与胚胎发育倒置相关的囊状肾脏的病理表现。我们在出生后一天获得纯合子小鼠，并将它们与野鼠进行比较。突变小鼠肾脏的一个显著特征是出现大小不一的管状囊。电子显微镜下可见扁平的上皮细胞呈立方体状，常无微血管和不规则形状的线粒体脱位。囊性小管内基底膜明显增厚。用凝集素双重染色区分肾单位段，囊变主要发生在远端小管。Na/K-ATPase亚基和Fodrin在基底侧均有染色，但在较大的囊肿内染色较弱。囊性小管内细胞角蛋白也呈弱阳性。总而言之，inv突变小鼠持续复制多晶体肾脏。由于候选基因编码的蛋白质涉及15个连续的Ankyrin基序，因此细胞骨架异常可能参与了肾脏结构异常、倒置和囊性形成的机制和产生。较少

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