CYSTIC DILATATION OF NEPHRONS IN TRANSGENIC INV MICE

转基因 INV 小鼠肾单位囊性扩张

• 批准号: 6262576
• 负责人: CARRIE L PHILLIPS
• 金额: $ 12.07万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6262576
• 关键词: autosomal recessive trait; chimeric proteins; developmental genetics; disease /disorder model; electron microscopy; embryogenesis; genetically modified animals; green fluorescent proteins; immunofluorescence technique; laboratory mouse; light microscopy; mammalian embryology; messenger RNA; polycystic kidney; postpartum; renal tubule

Polycystic kidney disease (PKD) is the most common inherited renal disease, accounting for 5-10% of end-stag renal disease with an annual cost of $240,000,000. The etiology of renal cysts is especially important for the children an adults with inherited PKD who may require dialysis or transplantation for survival. We have a newly describe transgenic mouse model of PKD, called inv/inv, which will be used to conduct studies not possible in humans. The long term goal of this project is to understand how the inheritance of a novel situs inversus gene, inv, results in PKD and loss of renal function. Our general strategy is to study the kidneys of inv/inv mice, which share phenotypic features with autosomal recessive (AR) PKD. They develop situs inversus, PKD and die prematurely from renal failure. The inv gene has been fully cloned and sequenced. Our preliminary data suggest inv is an intracellular protein with discrete localization in specific segments of the nephron. Our experiments will test the hypothesis that the inv gene codes for a developmentally important protein that maintains nephron integrity. Grant funding is a major factor in the PI's immediate goal of attaining research independence and the long-term career goal of becoming a successful and productive investigator in an academic institution. The specific aims of this present study are as follows: 1. To characterize the inv/inv mouse model of ARPKD, we will perform single and dual photon fluorescence microscopy with cell-specific antibodies and lectins. Immunogold electron microscopy with anti-inv antibodies will target the intracellular localization of the inv protein. These microscopy experiments will allow us to appropriately direct mechanistic studies of inv protein function and interactions. 2. In situ hybridization studies will be used to determine the cellular levels of inv messenger RNA during critical periods of development. This is especially important, as incremental variations in mRNA expression are known to occur during narrow developmental windows. 3. We will use green fluorescent protein-inv constructs and anti-inv antibodies t establish spatial and temporal inv expression patterns during development. We believe the inv/inv model offers a new an exciting opportunity to extend the understanding of the genetic mechanisms of nephrogenesis and the abnormalities in this process which lead to PKD.

多囊肾病（PKD）是最常见的遗传性肾病，占终末期肾病的5-10%，每年的费用为2.4亿美元。肾囊肿的病因是特别重要的儿童和成人遗传性PKD谁可能需要透析或移植的生存。我们有一个新描述的PKD转基因小鼠模型，称为inv/inv，它将用于进行在人类中不可能进行的研究。该项目的长期目标是了解一种新的反位基因inv的遗传如何导致PKD和肾功能丧失。我们的总体策略是研究inv/inv小鼠的肾脏，其与常染色体隐性（AR）PKD具有共同的表型特征。他们发展为内脏逆位、PKD并因肾衰竭而过早死亡。inv基因已被完全克隆和测序。我们的初步数据表明inv是一种细胞内蛋白，在肾单位的特定节段中具有离散的定位。我们的实验将验证inv基因编码一种在发育中重要的蛋白质的假设，这种蛋白质维持肾单位的完整性。补助资金是PI实现研究独立性的近期目标和成为学术机构中成功和富有成效的调查员的长期职业目标的主要因素。本研究的具体目的如下：1.为了表征ARPKD的inv/inv小鼠模型，我们将使用细胞特异性抗体和凝集素进行单光子和双光子荧光显微镜检查。使用抗inv抗体的免疫金电子显微镜将靶向inv蛋白的细胞内定位。这些显微镜实验将使我们能够适当地指导inv蛋白质功能和相互作用的机制研究。2.原位杂交研究将用于确定发育关键时期inv信使RNA的细胞水平。这是特别重要的，因为已知mRNA表达的增量变化发生在狭窄的发育窗口期间。3.我们将使用绿色荧光蛋白-inv构建体和抗inv抗体来建立发育期间inv的空间和时间表达模式。我们相信inv/inv模型提供了一个新的一个令人兴奋的机会，以扩大对肾发生的遗传机制和在这一过程中导致PKD的异常的理解。