Analysis of ARPKD by Targeted Manipulation of Pkhd1

通过 Pkhd1 的靶向操作分析 ARPKD

• 批准号: 7027118
• 负责人: CHRISTOPHER J WARD
• 金额: $ 28.52万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7027118
• 关键词: autosomal recessive trait; functional /structural genomics; gene deletion mutation; gene expression; gene induction /repression; gene targeting; genetic markers; genetic regulation; genetically modified animals; homozygote; laboratory mouse; membrane proteins; messenger RNA; phenotype; polycystic kidney; polymerase chain reaction; protein engineering; protein localization; protein quantitation /detection; protein structure function; scanning electron microscopy; secretory protein; spliceosomes; terminal nick end labeling; western blottings

DESCRIPTION (provided by applicant): Autosomal Recessive Polycystic Kidney Disease (ARPKD) is a devastating inherited neonatal nephropathy characterized by fusiform collecting duct dilatation and congenital hepatic fibrosis. We recently identified the ARPKD gene, PKHD1, and characterized a rat model of the disease, PCK. The PKHD1 gene is very large (477kb) generating a approximately 16kb mRNA transcribed from 67 exons. The ARPKD protein, fibrocystin, is a large (446kDa) integral membrane protein of unknown function. PKHD1 and the murine ortholog, Pkhd1, are thought to generate multiple splice forms, including possible secreted proteins. Mutation to PKHD1 is associated with a wide range of phenotypes from in utero presentation with greatly enlarged kidneys to hepatic disease only detected in adulthood. Mutation analysis has shown that most patients are compound heterozygotes for PKHD1 mutations and that patients with two truncating changes all have a severe renal phenotype resulting in perinatal death. This proposal is to target murine Pkhd1, to help determine the normal role of the protein and the consequences of various mutations. A targeted removal of exon 2 (Pkhd1 del2) has been engineered and homozygous animals have severe liver and pancreatic disease, but interestingly few renal cysts. Specific Aim 1 will characterize the phenotype and expression in these Pkhd1 del2 homozygotes. The possible influence of the incorporated neo cassette will be tested by floxing out by Cre expression and phenotypic reexamination. If the disease phenotype in the Pkhd1 del2(-neo) homozygotes remains mild further targeted disruption will be designed to generate a more severe renal phenotype: To test the role of the protein after renal and hepatic development, Specific Aim 2 will generate an inducible conditional knockout that will allow Pkhd1 to be mutated in somatic tissue and result in the elimination of fibrocystin in the adult or the neonate. The final set of experiments (Specific Aim 3) will tag the endogenous gene so that the protein can be localized and studied using reliable tag antibodies. Overall these studies should reveal more about the control of expression of Pkhd1, the normal role of fibrocystin and help clarify the mutational mechanism in this complex disorder. These are essential prerequisites before rational therapies can be developed for this disorder.

描述（申请人提供）：常染色体隐性多囊肾病（ARPKD）是一种破坏性的遗传性新生儿肾病，以梭状集管扩张和先天性肝纤维化为特征。我们最近鉴定了ARPKD基因PKHD1，并鉴定了该疾病的大鼠模型PCK。PKHD1基因非常大（477kb），产生大约16kb的mRNA，由67个外显子转录。ARPKD蛋白，纤维囊蛋白，是一个功能未知的大的（446kDa）完整膜蛋白。PKHD1和小鼠同源基因PKHD1被认为产生多种剪接形式，包括可能的分泌蛋白。PKHD1突变与多种表型相关，从子宫内肾脏肿大到仅在成年期检测到的肝脏疾病。突变分析表明，大多数患者是PKHD1突变的复合杂合子，有两个截断变化的患者都有严重的肾脏表型，导致围产期死亡。这项建议是针对小鼠Pkhd1，以帮助确定蛋白质的正常作用和各种突变的后果。外显子2 （Pkhd1 del2）的靶向去除已经被工程化，纯合子动物有严重的肝脏和胰腺疾病，但有趣的是很少有肾囊肿。特异性Aim 1将表征这些Pkhd1 del2纯合子的表型和表达。我们将通过Cre表达和表型复核来检测合并的新卡带可能产生的影响。如果Pkhd1 del2（-neo）纯合子的疾病表型仍然轻微，则进一步的靶向破坏将被设计为产生更严重的肾脏表型：为了测试该蛋白在肾脏和肝脏发育后的作用，Specific Aim 2将产生一个可诱导的条件敲除，使Pkhd1在体细胞组织中发生突变，并导致成人或新生儿中纤维囊蛋白的消除。最后一组实验（Specific Aim 3）将标记内源性基因，以便使用可靠的标记抗体对蛋白质进行定位和研究。总的来说，这些研究将揭示更多关于Pkhd1表达的控制，纤维囊素的正常作用，并有助于阐明这种复杂疾病的突变机制。这些都是为这种疾病开发合理疗法之前必不可少的先决条件。