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CYSTIC DILATATION OF NEPHRONS IN TRANSGENIC INV MICE

转基因 INV 小鼠肾单位囊性扩张

基本信息

批准号：
6899278
负责人：
CARRIE L PHILLIPS
金额：
$ 12.91万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-07-01 至 2007-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6899278
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 nephrogenesis polycystic kidney postpartum renal tubule

项目摘要

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脏疾病，占终末期肾脏疾病的5-10%，每年的费用为2.4亿美元。对于患有遗传性PKD的儿童和成人来说，肾囊肿的病因学尤为重要，因为他们可能需要透析或移植才能生存。我们有一个新的描述转基因小鼠PKD模型，称为inv/inv，它将用于进行不可能在人类身上进行的研究。该项目的长期目标是了解一种新的反向位置基因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与常染色体隐性（AR） PKD具有相同表型特征的inv/inv小鼠的肾脏。他们会出现逆位性肾脏病，并因肾功能衰竭而过早死亡。该基因已被完全克隆并测序。我们的初步数据表明inv是一种细胞内蛋白，在肾元的特定片段中具有离散的定位。我们的实验将验证一个假设，即inv基因编码一种维持肾元完整性的重要蛋白质。资助是PI实现研究独立的近期目标和成为学术机构中成功和富有成效的研究者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。免疫金电子显微镜与抗inv抗体将针对inv蛋白的细胞内定位。这些显微实验将使我们能够适当地指导inv蛋白功能和相互作用的机制研究。2. 原位杂交研究将用于确定在发育的关键时期的细胞水平的内向信使RNA。这一点尤其重要，因为已知mRNA表达的增量变化发生在狭窄的发育窗口期。3. 我们将使用绿色荧光蛋白inv构建体和抗inv抗体来建立发育过程中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令人兴奋的机会来扩展对肾发生的遗传机制和导致PKD的这一过程中的异常的理解。