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

常染色体显性多囊肾病（ADPKD）是最常见的遗传性多囊肾病。ADPKD的两个负责基因PKD1和PKD2已经被确定。为进一步了解ADPKD的发病机制和致病机制，我们拟建立以小鼠pkd1基因为靶点的ADPKD疾病模型。我们克隆了小鼠pkd1基因的全长cDNA。将亚克隆到GFP表达载体上的基因转染MDCK细胞系，用抗GFP抗体进行Western blotting证实蛋白产物的表达。虽然我们尝试制作pkd1转基因小鼠，但由于其基因的大尺寸，仍然没有成功。由于pkd1的转基因小鼠是四环素调控表达系统所必需的，我们的策略应该改变为制作其他类型的疾病模型。我们决定分两步来生成疾病模型。首先，将pkd1基因替换为外显子2的GFP和新霉素耐药基因的靶向载体转染到小鼠胚胎干细胞（ES）细胞系中。培养了pkd1基因敲除小鼠的杂合子。我们现在正在生成小鼠系。其次，为了产生双敲除嵌合体小鼠，应该在已经靶向一个等位基因的胚胎干细胞系中靶向另一个等位基因。下一步是通过将pkd1基因替换为2外显子GFP和潮霉素耐药基因的靶向载体，筛选重组ES细胞系。这种小鼠由两种细胞组成。一种细胞类型的pkd1为零突变，另一种细胞类型的pkd1为正常突变。因此，可以预期生成的小鼠与人类ADPKD表型相似。对pkd1敲除小鼠和双敲除嵌合体小鼠的纯合子进行比较分析，将有助于揭示ADPKD的发病机制和致病机制。

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.