Generation of the disease-model mice for autosomal dominant polycystic kidney disease using tetracycline regulatory expression systems

使用四环素调节表达系统产生常染色体显性多囊肾病疾病模型小鼠

• 批准号: 13671093
• 负责人: MOCHIZUKI Toshio
• 金额: $ 2.62万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671093/
• 关键词: Polycystic kidney diseasee; PKD gene; knock-out mice; テトラサイクリン制御性発現系; 常染色体優性多発性嚢胞腎; ADPKD; PKD1; ポリシスチン; コンディショナルノックアウトマウス

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary cystic renal disease. Two responsible genes for ADPKD, PKD1 and PKD2, have been identified. Here we set out to develop the disease- model of ADPKD, which is targeted mouse pkd1 gene, for understanding the pathogenesis and the disease-causing mechanism of ADPKD.We have cloned the full-length cDNA of mouse pkd1 gene. The gene subcloned into GFP expression vector was transfected into MDCK cell line and the expression of the protein product was confirmed by Western blotting using anti-GFP antibody. Although we tried to make pkd1 transgenic mice, because of its large size of the gene, it is still unsuccessful.Since transgenic mice for pkd1 are necessary for tetracycline regulatory expression systems, our strategy should be changed to make other types of disease-model. We decided take two steps to generate the disease-model.First, the targeting vector, which is replaced pkd1 gene to GFP and neomycin resistant genes in exon 2, have been transfected into mice embryo stem (ES) cell line. Heterozygotes of knock-out mice for pkd1 gene were developed. We are now generating the mice line.Second, in order to generate double knock-out chimera mice, another allele should be targeted in ES cell line which has already been targeted one allele. The next step is screening of ES cell line which is recombinant by targeting vector which is replaced pkd1 gene to GFP and hygromycin resistant genes in exon 2. This mice consist of two cell types. One cell type has null mutation for pkd1 and another has normal for pkd1. Therefore, the generated mice could be expected to resemble human ADPKD phenotype. Comparative analysis of homozygotes for pkd1 knock-out mice and double knock-out chimera mice will be shed light on the pathogenesis and the disease-causing mechanism of ADPKD.

常染色体显性多囊肾病（ADPKD）是最常见的遗传性囊性肾病。已鉴定出ADPKD的两个负责基因PKD1和PKD2。本研究以小鼠pkd 1基因为靶基因，建立了ADPKD的动物模型，并克隆了小鼠pkd 1基因的全长cDNA。将亚克隆到GFP表达载体中的基因转染MDCK细胞系，并使用抗GFP抗体通过Western印迹证实蛋白产物的表达。虽然我们尝试过制备pkd 1转基因小鼠，但由于其基因体积较大，目前仍不成功，由于四环素调控表达系统需要pkd 1转基因小鼠，因此我们的策略应改为制备其他类型的疾病模型。我们决定分两步建立疾病模型：首先，将pkd 1基因替换为GFP基因，并在第2外显子插入新霉素抗性基因的靶向载体转染小鼠胚胎干细胞（ES细胞）。建立了pkd 1基因敲除小鼠的杂合子。第二，为了产生双敲除嵌合体小鼠，需要在已经靶向一个等位基因的ES细胞系中靶向另一个等位基因。下一步是筛选用靶向载体将pkd 1基因替换为GFP和潮霉素抗性基因的ES细胞系。这种小鼠由两种细胞类型组成。一种细胞类型具有PKD1的无效突变，另一种细胞类型具有PKD1的正常突变。因此，预期生成的小鼠与人ADPKD表型相似。通过对pkd1基因敲除小鼠和双基因敲除嵌合体小鼠纯合子的比较分析，将有助于进一步阐明ADPKD的发病机制。