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

• Project/Area Number: 13671093
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Kidney internal medicine
• Research Institution: HOKKAIDO UNIVERSITY
• Principal Investigator: MOCHIZUKI Toshio Hokkaido Univ., Grad., School of Medicine., Assist. Prof., 大学院・医学研究科, 講師 (00277120)
• Project Period (FY): 2001 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥4,100,000 (Direct Cost: ¥4,100,000); Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000); Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
• Keywords: 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

Research Abstract

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.