| Research Abstract |
We recently cloned a new protein, parchorin, from rabbit choroid plexus. Parchorin belongs to Chloride Intracellular Channel (CLIC) family and is specifically expressed in cells participating water movement in various tissues. In the present study, we tried to produce a parchorin-knock out mouse to utilize it as a disease model. Based on its rabbit sequence, RT-PCR was performed for mRNA obtained from mouse choroid plexus. Although we cloned a new family member of CLIC, it did not seem to be parchorin. Meanwhile, one of our colleague isolated human CLIC5A and its splicing variant CLIC5B. From its sequence and tissue distribution, our cloned sequence was considered to be a mouse homologue of CLIC5A. We then purified parchorin protein to get amino acid sequence, and determined the whole sequence of mouse parchorin utilizing, the results of mouse genome project as well. Based on the published genomic data, construction of targeting vector for substituting parchorin exon 1 was performed. We employed a strategy to eliminate neomycin resistant gene for selection as well as to enable a conditional knock out using both Cre/lox and FLP/Frt systems. Using established methods, recombinant mouse ES cells and subsequently chimera mice were obtained. At the end of the project, hetero-mice were born. We are now planning to analyze their pathophysiology after the knockout mice are born.
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