权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Generation of aldose reductase knockout mouse by gene targeting and expression of human enzyme

通过基因打靶和人酶表达产生醛糖还原酶敲除小鼠

基本信息

批准号：
06670132
负责人：
NISHIMURA Chihiro
金额：
$ 1.34万
依托单位：
National Children's Medical Research Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670132/
关键词：
aldose reductase gene targeting diabetic complications 酵素欠失マウス

项目摘要

Aldose reductase (alditol : NAD(P)+ 1-oxidoreductase) is an enzyme implicated in the pathogenesis of various diabetic complications. To elucidate the physiological function as well as the molecular, mechanisms underlying the involvement of this enzyme in the development of diabetic complications, we planned to 1) generate the mice lacking the enzyme by gene targeting and to 2) express human enzyme in these mice.To construct targeting vectors to knockout aldose reductase (AR) gene, genomic fragments of mouse AR gene was required. Among the initial 25 clones obtained from mouse genomic library, none of the clones was identical to the mouse vas deferens protein, previously reported as the mouse counterpart of AR. We therefore newly screened the mouse kidney cDNA Iibrary using rat cDNA probe. The isolated clone encoded a 316-amino acid protein with 97 % identity to rat lens AR and 69 % identity to the mouse vas deferens protein. RNA blot analysis demonstrated abundant expression of the enz … More yme transcript in the testis, skeletal muscle and kidney. The isolated cDNA was expressed in Escherichia coli and the recombinant protein was purified to homogeneity by affinity chromatography and chromatofocusing. The expressed enzyme demonstrated the typical characteristics of AR previously reported for the enzyme protein isolated from other animal species. These results indicated the presence of a closely related subgroup within the aldo-keto reductase superfamily in mouse tissues. Based on the sequence data obtained from the cDNA clone, PCR primers were synthesized to identify the genomic clone of mouse enzyme among the former 25 isolated clones. Two of the clones were demonstrated to contain intron structure. Analysis of the restriction enzyme map and the sequences of these genomic clones indicated that mouse AR gene spans approximately 14 kb and encoded by 10 exons. The boundaries between exons and introns are determined by comparing the genomic sequence to cDNA sequence. All of the splice junctions of the gene conformed with the GT splice donor and AG splice acceptor rule. Presently we are making targeting vectors to generate knockout mice by use of the genomic fragments isolated from this gene. Less

醛糖还原酶（alditol：NAD（P）+1-oxidoreductase）是一种与各种糖尿病并发症的发病机制有关的酶。为了阐明该酶在糖尿病并发症发生中的生理功能和分子机制，我们计划1）通过基因打靶方法产生该酶缺失的小鼠，2）在这些小鼠中表达人源性的酶，构建敲除小鼠醛糖还原酶（aldose reductase，AR）基因的靶向载体，需要小鼠AR基因的基因组片段。在从小鼠基因组文库获得的最初25个克隆中，没有一个克隆与先前报道为小鼠AR对应物的小鼠输精管蛋白相同。因此，我们用大鼠cDNA探针新筛选了小鼠肾cDNA文库。分离的克隆编码316个氨基酸的蛋白质，与大鼠透镜AR具有97%的同一性，与小鼠输精管蛋白具有69%的同一性。RNA印迹分析表明，该酶的表达丰富， ...更多信息在睾丸、骨骼肌和肾脏中的YME转录物。在大肠杆菌中表达重组蛋白，并通过亲和层析和层析聚焦纯化。表达的酶表现出AR的典型特征，以前报道的酶蛋白分离自其他动物物种。这些结果表明在小鼠组织中醛酮还原酶超家族中存在密切相关的亚组。根据获得的cDNA克隆的序列数据，合成PCR引物以鉴定前25个分离的克隆中的小鼠酶基因组克隆。两个克隆被证明含有内含子结构。对这些克隆的限制性内切酶图谱和基因组序列的分析表明，小鼠AR基因跨度约为14 kb，由10个外显子编码。外显子和内含子之间的边界通过比较基因组序列和cDNA序列来确定。该基因的所有剪接点均符合GT剪接供体和AG剪接受体规则。目前，我们正在利用从该基因分离的基因组片段制备靶向载体以产生敲除小鼠。少