Combined BAC Transgenic and Knock-Out Mouse Model of Lowe Syndrome Nephropathy

Lowe 综合征肾病的 BAC 转基因和敲除小鼠模型相结合

基本信息

批准号：
8627601
负责人：
ROBERT L NUSSBAUM
金额：
$ 33.6万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8627601
关键词：
Accounting Address Affect American Society of Hematology Amino Acids Animals Anxiety Apical Backcrossings Bacterial Artificial Chromosomes Behavioral Binding Brain Cataract Cell membrane Cells Chloride Channels Clathrin Complex Congenic Mice Defect Disease Embryo Endocytosis Endosomes Enzymes Epitopes Exons Financial compensation Functional disorder Generations Genes Genetic Recombination Genetic Transcription Germ Cells Golgi Apparatus Growth Homologous Gene Human Human Genome Immunoprecipitation Kidney Kidney Diseases Knock-out Knockout Mice LDL-Receptor Related Protein 2 Learning Link Mediating Memory Memory impairment Mental Retardation Metabolic Pathway Metabolism Molecular Weight Motor Mouse Strains Mus Mutant Strains Mice Mutation Neuraxis Neurologic Oculocerebrorenal Syndrome PH Domain Patients Peptides Phenotype Phosphatidylinositol Phosphates Phosphatidylinositols Phospholipids Phosphoric Monoester Hydrolases Proteins Proteinuria Proximal Kidney Tubules RNA Splicing Rare Diseases Recycling Research Proposals Structure Testing Transgenic Mice Transgenic Organisms Tubular formation Urine base coated pit cohort congenital cataract disease-causing mutation embryo tissue embryonic stem cell enzyme activity homologous recombination human tissue insight lens loss of function mutation motor deficit mouse genome mouse model promoter receptor rho stem trafficking trans-Golgi Network yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): The Lowe OculoCerebroRenal syndrome of (LOCR) is a rare X-linked disorder of congenital cataracts, mental retardation, behavioral abnormalities, and renal tubulopathy. The disease is caused by loss-of-function mutations in the OCRL gene, which encodes Ocrl, a phosphatidylinositol(4,5)P2 5-phosphatase located in the trans-Golgi network, endosomes, plasma membrane ruffles, and clathrin-coated pits. The renal tubular abnormalities of LOCR, particularly low molecular weight (LMW) proteinuria and aminoaciduria, overlap with those of Dent disease, a disorder that affects only the kidney. Mutations in OCRL account for ~20% of Dent disease patients. It has been proposed that derangements in phosphatidylinositol(4,5)P2 metabolism in LOCR causes defective receptor endocytosis, such as apical trafficking or recycling of megalin, a protein responsible for reabsorbing LMW proteins in the kidney proximal tubule. The CNS abnormalities could arise by analogous defects in recycling of certain CNS receptors. Testing these hypotheses is hampered by the lack of a mouse model because mice with a knock-out of Ocrl, the urine homolog of OCRL, have no detectable phenotype. We have shown that the lack of renal phenotype in Ocrl-knockout mice is due to complete compensation by another phosphatidylinositol(4,5)P2 5-phosphatase, Inpp5b, encoded by murine Inpp5b and mouse INPP5B. Mice with a mutation in Inpp5b have only a mild phenotype but mutation of both Ocrl and Inpp5b causes early embryonic lethality, indicating functional overlap. INPP5B and Inpp5b have clear differences in transcription and splicing which could explain why INPP5B only partially compensates for loss of human OCRL while mouse Inpp5b completely compensates. We created transgenic mice deleted for both murine Ocrl and Inpp5b and hemizygous for a bacterial artificial chromosome (BAC) containing INPP5B, and found they have delayed growth and the proximal renal tubular defects seen in LOCR and Dent disease. Mice deleted for both murine Ocrl and Inpp5b and homozygous for the INPP5B-BAC insertion grow more normally and excrete much less LMW protein. We conclude that there is both a qualitative and quantitative difference in the ability of INPP5B and Inpp5b to compensate for loss of Ocrl in mice. In this current application, we propose to culture proximal tubular cells from these strains of mice and correlate INPP5B enzyme activity with subcelular levels of phosphatidylinositol(4,5)P2 and altered renal transport of low molecular weight proteins and amino acids. We will also use recombination in mouse embryonic stem cells to dissect and identify the differences between INPP5B and Inpp5b responsible for the difference in phenotype between Ocrl-deficient humans and mice. Finally, after completing a 10-generation backcross onto C57Bl/6, we will examine the mice for abnormalities in central nervous system by testing motor function, anxiety, learning, memory and by examining the brains neuropathologically.

描述（由申请人提供）：（LOCR）的Lowe眼脑综合征是一种罕见的先天性白内障，智力低下，行为异常和肾小管病的X连锁疾病。该疾病是由OCRL基因中的功能丧失突变引起的，该基因编码位于反式高尔基网络中的OCRL，磷脂酰肌醇（4,5）P2 5-磷酸酶，内体，质膜膜和clathrin涂有蛋白的pits。 LOCR的肾小管异常，尤其是低分子量（LMW）蛋白尿和氨基酸尿症，与Dent病的肾小管异常重叠，这种疾病仅影响肾脏。 OCRL中的突变占凹陷疾病患者的约20％。已经提出，LOCR中磷脂酰肌醇（4,5）P2代谢的扰动会导致受体内吞作用有缺陷，例如顶端运输或梅加林的再生蛋白质（一种负责在肾脏近前小管中取得LMW蛋白质的蛋白质）的蛋白质。中枢神经系统异常可能是由于某些中枢神经系统受体回收的类似缺陷而引起的。缺乏小鼠模型来测试这些假设会阻碍，因为OCRL（OCRL的尿液同源物）的小鼠没有可检测的表型。我们已经表明，OCRL-敲除小鼠中缺乏肾脏表型是由另一种磷脂酰肌醇（4,5）P2 5-磷酸酶（INPP5B）完全补偿，由Mirine INPP5B和小鼠INPP5B编码。 Inpp5b中突变的小鼠仅具有轻度的表型，但是OCRL和INPP5B的突变会引起早期的胚胎致死性，表明功能重叠。 INPP5B和INPP5B在转录和剪接方面存在明显的差异，这可以解释为什么INPP5B仅部分补偿人OCRL的损失，而小鼠INPP5B完全补偿了人类OCRL。我们创建了针对鼠OCRL和INPP5B删除的转基因小鼠，并为含有Inpp5b的细菌人造染色体（BAC）删除，并发现它们延迟了生长，并且在LOCR和DENT疾病中看到的近端肾小管缺陷。为鼠OCRL和INPP5B删除的小鼠和INPP5B-BAC插入的纯合生长的生长更为正常，而LMW蛋白的排泄更少。我们得出的结论是，INPP5B和INPP5B能够补偿小鼠OCRL损失的能力既存在定性和定量差异。在当前的应用中，我们建议从这些小鼠菌株中培养近端管状细胞，并将Inpp5b酶活性与亚丝网磷脂酰肌醇（4,5）P2相关，并改变了低分子量蛋白和氨基酸的肾脏转运。我们还将在小鼠胚胎干细胞中使用重组，以剖析和识别inpp5b和Inpp5b之间的差异，以使OCRL缺乏人类和小鼠之间的表型差异。最后，在完成C57BL/6上的10代反跨后，我们将通过测试运动功能，焦虑，学习，记忆和通过神经病理学检查大脑的运动功能，焦虑，学习，记忆，检查中枢神经系统中的异常。