A Potential Zebrafish Model of Glaucoma by Genetic Ablation and Modification of t

通过遗传消融和修饰来建立潜在的青光眼斑马鱼模型

基本信息

批准号：
7360019
负责人：
XINPING Charlie ZHAO
金额：
$ 21.84万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-12-01 至 2009-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7360019
关键词：
Ablation Address Adult Affect Animal Model Animals Anterior Apoptosis Appearance Aqueous Humor Blindness CB1954 Cell Aggregation Cell Count Cell Death Cells Characteristics Chemicals Closure Clutch Size Cornea Corynebacterium diphtheriae Cytotoxic agent Development Diphtheria Toxin Disease Disruption Dominant-Negative Mutation Drainage procedure Dropout E coli NfsA protein Elevation Embryo Etiology Excision Eye Eye diseases Fishes Fresh Water Functional disorder Future Gene Expression Genes Genetic Genetic Models Genetic Structures Genome Genomics Glaucoma Human Individual Iris Knowledge Lead Life Cycle Stages Ligaments Liquid substance Localized Maintenance Mammals Measurement Measures Methods Metronidazole Minimally Invasive Surgical Procedures Modeling Modification Molecular Molecular Genetics Morphology Mutate Mutation Nitroreductases Numbers Obstruction Optic Nerve Pathway interactions Patients Pharmaceutical Preparations Pharmacotherapy Physiologic Intraocular Pressure Physiological Play Preclinical Drug Evaluation Process Prodrugs Protein Biosynthesis Protein Overexpression Proteins Range Regulation Resources Retinal Ganglion Cells Risk Factors Role Shapes Structure Symptoms System Testing Tissues Trabecular meshwork structure Transgenic Organisms Variant Vision Visual Fields Zebrafish analytical method cytotoxic disease mechanisms study gain of function ganglion cell genetic analysis genetic manipulation glaucoma test human disease mutant myocilin optic nerve disorder positional cloning pressure prevent promoter size therapy development tool

项目摘要

DESCRIPTION (provided by applicant): Glaucoma is a group of heterogeneous disorders characterized by optic neuropathies, dropout of retinal ganglion cells, and visual field loss. It affects an estimated 70 million people and is the second leading cause of blindness worldwide. The exact mechanisms through which glaucoma occurs are not clearly known. Current treatments are far from satisfactory. The objective of our studies is to understand the physiopathology of glaucoma at the cellular and molecular levels and use this knowledge to develop future therapies to prevent and treat this significant blinding disease. This application explores the possibility of developing a genetic model of glaucoma in zebrafish using genetic and molecular tools. The zebrafish (Danio rerio) has become a leading model animal for the studies of human diseases because it allows powerful forward and reverse genetic analysis to be performed to quickly identify genetic loci and study their roles in the disease process. Anatomical, physiological, and genetic conservation of the important eye tissues that play key roles in maintaining intraocular pressure (IOP) in zebrafish and humans strongly suggests that the fish is a promising model animal for studying glaucoma. The annular ligament (AL) in the zebrafish eye is a specialized structure that is anatomically localized in the eye where the mammalian trabecular meshwork (TM) resides. Because TM is the primary tissue in regulation IOP, a key risk factor for glaucoma, zebrafish may develop glaucoma or glaucoma-like symptoms if its normal structure of the AL is altered by genetic ablation or modification of the AL cells. To test this hypothesis, transgenic fish will be produced by inserting three types of genes into the fish genome: a toxic gene (diphtheria toxin A, DTA), the E. coli nitroreductase (NTR) gene whose protein converts a harmless chemical compound (prodrug) into cytotoxic species, and the mutant myocilin gene that is causative for glaucoma and is known to alter the TM cells. Expression of these genes in the AL will lead to cell death or aggregation, which may result in obstruction of the aqueous humor outflow and elevated IOP. Tissue-specific cell ablation and modification is controlled by the AL-specific promoter and an inducible cre/loxP system. Histological, physiological, immunohistochemical, and molecular methods will be used to evaluate what happens to the transgenic fish in terms of overall structure of the eye, morphology in the AL, changes in IOP, cell number and viability in retinal ganglion cells, and appearance of optic nerve. The transgenic zebrafish developed in this proposal may mimic human glaucoma and provide an invaluable tool for further studying the etiology of glaucoma and for developing new drug as an affordable and high throughput system. The zebrafish (Danio rerio), a small fresh water fish and a popular model animal for the studies of human diseases, is genetically modified by selective removal or alteration of cells important to regulate normal intraocular pressure (IOP), a measurement of the fluid pressure inside the eye that helps maintain the shape of the eye. In glaucoma patients, IOP is often elevated, leading to irreversible vision loss. The fish generated in this study may have a raised IOP and develop glaucoma, providing an affordable and high throughput system for glaucoma drug and therapy development.

描述（由申请人提供）：青光眼是一组以视神经病，视网膜神经节细胞辍学和视野损失为特征的异质性疾病。它影响了估计的7000万人，是全球失明的第二大主要原因。青光眼发生的确切机制尚不清楚。目前的治疗远非令人满意。我们研究的目的是了解细胞和分子水平上青光眼的生理病理学，并利用这些知识来开发未来的疗法，以预防和治疗这种严重的盲目疾病。该应用探讨了使用遗传和分子工具在斑马鱼中开发青光眼遗传模型的可能性。斑马鱼（Danio Rerio）已成为人类疾病研究的主要模型动物，因为它允许进行强大的前进和逆转遗传分析，以快速识别遗传基因座并研究其在疾病过程中的作用。重要眼组织的解剖学，生理和遗传保护在斑马鱼和人类中保持眼内压（IOP）方面起着关键作用，这强烈表明，鱼是研究青光眼的有前途的模型动物。斑马鱼眼中的环形韧带（Al）是一种专业结构，在哺乳动物小梁网（TM）驻留在眼睛中的解剖结构。由于TM是调节IOP中的主要组织，这是青光眼的关键危险因素，斑马鱼可能会出现青光眼或青光眼样症状，如果其Al的正常结构通过AL细胞的遗传消融或修饰而改变。为了检验这一假设，将通过将三种类型的基因插入鱼类基因组中产生转基因鱼类：毒性基因（白喉毒素A，DTA），大肠杆菌硝酸盐质合酶（NTR）基因的蛋白质，其蛋白质会转化无害化合物（Prodrug））进入细胞毒性物种和对青光眼致病的突变肌动蛋白基因，众所周知会改变TM细胞。这些基因在AL中的表达将导致细胞死亡或聚集，这可能导致幽默流出和IOP升高。组织特异性的细胞消融和修饰由Al特异性启动子和可诱导的CRE/LOXP系统控制。组织学，生理，免疫组织化学和分子方法将使用眼睛的整体结构，AL中的形态，IOP的变化，视网膜神经节细胞中的细胞数和生存能力以及视网膜细胞中的细胞数和生存能力评估转基因鱼会发生什么以及视神经。该提案中开发的转基因斑马鱼可能模仿人类青光眼，并为进一步研究青光眼的病因和开发新药物作为一种负担得起的高通量系统提供了宝贵的工具。斑马鱼（Danio Rerio）是一种小的淡水鱼类和一种流行的人类疾病研究模型动物，通过选择性去除或对调节正常内压（IOP）的细胞的改变或改变的细胞的变化在遗传上修饰，这是对流体压力的测量眼睛内部有助于保持眼睛的形状。在青光眼患者中，IOP通常会升高，导致视力丧失。这项研究中产生的鱼可能会增加IOP并发展青光眼，为青光眼药物和治疗开发提供了负担得起的高通量系统。