Development of a disease model targeting on a newly discovered protein, parchorin, specifically expressed in water-secreting cells

我们最近从兔的脉络丛中克隆了一种新的蛋白质Parclin。Parclin属于氯离子细胞内通道(CLIC)家族，在多种组织中参与水分运动的细胞中特异表达。在目前的研究中，我们试图制造一种Parclin基因敲除的小鼠来作为疾病模型。根据ITS兔序列，对从小鼠脉络丛中获得的mRNA进行RT-PCR。虽然我们克隆了一个新的CLIC家族成员，但它似乎不是parclin。同时，我们的一位同事分离到了人类CLIC5A及其剪接变异体CLIC5B。从ITS序列和组织分布来看，我们克隆的序列可能是CLIC5A的小鼠同源物。然后提纯Parclin蛋白，得到氨基酸序列，并利用小鼠基因组计划的结果测定了小鼠Parclin的全序列。在已发表的基因组数据的基础上，构建了取代parclin外显子1的靶向载体。我们采用了一种策略来消除新霉素抗性基因进行选择，并使用Cre/lox和FLP/FRT系统进行条件敲除。利用已建立的方法，获得了重组小鼠ES细胞和随后的嵌合体小鼠。在项目结束时，异种小鼠诞生了。我们现在正计划在基因敲除小鼠出生后分析它们的病理生理学。

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.