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Functional knockout of the glucose transporter 2 in mice overexpressing a dominant negative mutation

过度表达显性失活突变的小鼠中葡萄糖转运蛋白 2 的功能性敲除

基本信息

批准号：
14570716
负责人：
OHURA Toshihiro
金额：
$ 2.5万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Fanconi-Bickel syndrome (FBS) is an autosomal recessive disorder characterized by hepatorenal glycogen accumulation, Fanconi nephropathy and impaired utilization of glucose and galactose. Recently several mutations in GLUT2 gene were reported in FBS patients. We performed molecular analysis of three Japanese patients and found four novel mutations : a splice-site mutation (IVS2-2A>G), a nonsense mutation (Q287X) and two missense mutations (L389P and V423E). The family members who presented renal glucosuria were heterozygous for V423E mutation. If some mutant GLUT2 proteins such as V423E have a dominant-negative effect, an oligomer composed of mutant and wild-type proteins could result in abolition of transport activity.In this experiment, we tried to create model mouse for Fanconi-Bickel syndrome using functional knockout by overexpression of dominant negative inhibitor. The expression vector consists of the CAG promoter, a loxP, the DsRed, a second loxP, the mutant GLUT2 cDNA (V423E), the IRES, and the EGFP, in that order. The CDRE-GLUT2 unit was excised and microinjected into fertilized mouse eggs. We finally obtained 4 different strains, which over expressed mutant GLUT2 cDNA. We measured the blood glucose levels of these mice and checked the glucosuria but there were no significant difference between control mice and transgenic mice. We speculated that expression of mutant GLUT2 in transgenic mice was not enough to produce functional knockout or that V423E mutation had not dominant-negative effects.

Fanconi-Bickel综合征（FBS）是一种常染色体隐性遗传疾病，以肝肾糖原累积、Fanconi肾病和葡萄糖和半乳糖利用受损为特征。近年来，在FBS患者中发现了几种GLUT 2基因突变。我们对三名日本患者进行了分子分析，发现了四个新的突变：剪接位点突变（IVS 2 -2A>G），无义突变（Q287 X）和两个错义突变（L389 P和V423 E）。肾性糖尿的家系成员均为V423 E杂合子。如果某些突变的GLUT 2蛋白如V423 E具有显性负效应，则突变蛋白和野生型蛋白组成的寡聚体可导致转运活性的丧失。表达载体依次由CAG启动子、loxP、DsRed、第二loxP、突变体GLUT 2 cDNA（V423 E）、IRES和EGFP组成。切下CDRE-GLUT 2单位并显微注射到受精的小鼠卵中。最终获得了4株高表达突变型GLUT 2 cDNA的菌株。我们测量了这些小鼠的血糖水平并检查了糖尿，但在对照小鼠和转基因小鼠之间没有显着差异。我们推测突变型GLUT 2在转基因小鼠中的表达不足以产生功能性敲除或V423 E突变不具有显性负效应。