| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
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
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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
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