Analysis of ARPKD by Targeted Manipulation of Pkhd1

通过 Pkhd1 的靶向操作分析 ARPKD

• 批准号: 6859407
• 负责人: CHRISTOPHER J WARD
• 金额: $ 29.21万
• 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/6859407
• 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（由申请人提供）：常染色体隐性多囊肾病（ARPKD）是一种毁灭性的遗传性新生儿肾病，其特征是梭形集合管扩张和先天性肝纤维化。 我们最近确定了ARPKD基因PKHD 1，并描述了该疾病的大鼠模型PCK。 PKHD 1基因非常大（477 kb），产生从67个外显子转录的约16 kb mRNA。 ARPKD蛋白，纤维囊蛋白，是一个大的（446 kDa）的功能未知的膜整合蛋白。 PKHD 1和鼠直系同源物Pkhd 1被认为产生多种剪接形式，包括可能的分泌蛋白。 PKHD 1突变与从子宫内表现的肾脏极大增大到仅在成年期检测到的肝病的广泛表型相关。 突变分析表明，大多数患者是PKHD 1突变的复合杂合子，并且具有两个截短变化的患者都具有导致围产期死亡的严重肾脏表型。 该建议是针对鼠Pkhd 1，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（Pkhd 1 del 2）的靶向去除已经被工程化，纯合子动物具有严重的肝脏和胰腺疾病，但有趣的是很少有肾囊肿。 特异性目的1将表征这些Pkhd 1 del 2纯合子中的表型和表达。 将通过Cre表达和表型复检的floxing out来测试掺入的neo盒的可能影响。 如果Pkhd 1 del 2（-neo）纯合子中的疾病表型保持轻度，则将设计进一步的靶向破坏以产生更严重的肾脏表型：为了测试蛋白质在肾脏和肝脏发育后的作用，特异性目标2将产生诱导型条件性敲除，其将允许Pkhd 1在体细胞组织中突变，并导致成人或新生儿中纤维囊蛋白的消除。 最后一组实验（特定目标3）将标记内源基因，以便使用可靠的标签抗体定位和研究蛋白质。 总的来说，这些研究应该揭示更多关于Pkhd 1表达的控制，纤维囊蛋白的正常作用，并有助于澄清这种复杂疾病的突变机制。 这些都是必要的先决条件之前，合理的治疗方法可以为这种疾病的发展。