Alpha Crystallin Function in the Zebrafish

斑马鱼的α晶状体蛋白功能

• 批准号: 9021559
• 负责人: Mason Posner
• 金额: $ 30.56万
• 依托单位: ASHLAND UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9021559
• 关键词: 2 year old; Adopted; Aging; Amino Acids; Biochemical; Biochemistry; Biological Models; Biology; Blindness; Cataract; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complement; Confocal Microscopy; Crystallins; Data; Development; Disease; Embryo; Etiology; Family; Fishes; Funding; Gene Expression; Genes; Genome; Goals; Heat shock proteins; Human; Hydrophobicity; In Vitro; Knock-out; Laboratories; Laboratory Research; Lead; Lens Opacities; Lens development; Life; Maintenance; Mammals; Mass Spectrum Analysis; Measures; Methods; Modeling; Molecular; Molecular Chaperones; Monitor; Mutation; Oligonucleotides; Phenotype; Play; Population; Post-Translational Protein Processing; Prevention; Property; Proteins; Proteomics; Publishing; Reporting; Research Personnel; Role; Staging; Structure; Students; System; Techniques; Testing; Time; Training; Transgenic Mice; Universities; Variant; Vision; Vision research; Work; Zebrafish; age related; alpha-Crystallins; base; comparative; congenital cataract; cost efficient; experience; genome editing; in vitro Model; in vivo; in vivo Model; innovation; interest; lens; lens transparency; light microscopy; mouse model; mutant; novel; overexpression; paralogous gene; plasmid DNA; prevent; promoter; protein aggregation; protein expression; public health relevance; research study; tool; undergraduate research; university student; zebrafish development; zygote

 DESCRIPTION (provided by applicant): Alpha-crystallins are a family of small heat shock proteins that play a central role in lens development and the maintenance of lens transparency. Evidence suggests that α-crystallins prevent lens cataracts during aging, and that mutations in these proteins can lead to congenital cataracts. Previous work in our laboratory and in others shows that lens development, lens biochemistry and α-crystallin function are well conserved between mammals and zebrafish, supporting the use of zebrafish as a model system for lens biology. The goal of this current proposal is to develop a fast and cost efficient in vivo system fr assessing the functional properties of native and variant α-crystallins by leveraging recent tools for manipulating gene expression in the zebrafish. In Aim I, undergraduate research students will be trained in an innovative genome editing technique (called CRISPR/Cas) to disrupt the three zebrafish α- crystallin genes and produce genetically modified fish populations lacking combinations of each protein. A variety of morphological, histological and proteomic techniques will be used to assess the resulting impact of disrupting the function of α-crystallin genes. Changes in protein expression, aggregation and post-translational modification in each "functional knockout" fish population will be examined to identify the role of α-crystallins in les development and the modulation of age-related changes that can lead to cataract. The ability of zebrafish to produce large numbers of externally developing transparent embryos provides a powerful tool for identifying potential effects of α- crystallin loss from the earliest stages of ye development to old age (2 years). In Aim II, a zebrafish mutant strain with congenital cataracts will be used as an in vivo model system for assessing the ability of native and modified α-crystallins to prevent disease-causing protein aggregation. Students will construct DNA plasmids to drive the expression of specific α-crystallin genes and microinject them into single-celled zebrafish zygotes. Increases in α-crystallin expression will be quantified by targeted mass spectrometry and the ability of each expressed α- crystallin to prevent cataract will be assessed by Nomarski light microscopy and confocal microscopy. The in vivo zebrafish tools produced in this study will complement the use of mammalian models and in vitro techniques for analyzing the roles of α-crystallin in the lens. This work is relevant to vision researchers interested in te etiology and prevention of cataract, a leading cause of human blindness worldwide.

 描述(申请人提供)：α-晶体蛋白是一个小的热休克蛋白家族，在晶状体发育和维持晶状体透明度方面发挥核心作用。有证据表明，α-晶体蛋白可以防止晶状体老化过程中的白内障，而这些蛋白质的突变可能会导致先天性白内障。我们实验室和其他实验室的先前工作表明，哺乳动物和斑马鱼之间的晶状体发育、晶状体生物化学和α晶体蛋白功能非常保守，支持将斑马鱼用作晶状体生物学的模型系统。本提案的目标是利用最新的工具开发一种快速且经济高效的体内系统来评估天然的和变异的α晶体蛋白的功能特性 操纵斑马鱼的基因表达。在目标I中，本科生将接受一种创新的基因组编辑技术(称为CRISPR/CAS)的培训，以破坏三种斑马鱼α-晶体蛋白基因，并产生缺乏每种蛋白质组合的转基因鱼类种群。将使用各种形态、组织学和蛋白质组学技术来评估破坏α-晶体蛋白基因功能所产生的影响。我们将研究每个“功能敲除”鱼类种群中蛋白质表达、聚集和翻译后修饰的变化，以确定α-晶体蛋白在LES发育中的作用，以及可能导致白内障的年龄相关变化的调节。斑马鱼产生大量外部发育的透明胚胎的能力为确定从胚胎发育的最早阶段到老年(2岁)α-晶体蛋白丢失的潜在影响提供了一个强有力的工具。在AIM II中，一个患有先天性白内障的斑马鱼突变株将被用作体内模型系统，以评估天然和修饰的α晶体蛋白防止致病蛋白聚集的能力。学生们将构建dna质粒来驱动特定的α晶体蛋白基因的表达，并将它们显微注射到斑马鱼的单细胞受精卵中。α-晶体蛋白表达的增加将通过靶向质量来量化 每个表达的α晶体蛋白的光谱和预防白内障的能力将通过诺马尔斯基光学显微镜和共聚焦显微镜进行评估。这项研究中产生的活体斑马鱼工具将补充哺乳动物模型和体外技术的使用，以分析晶状体中α晶体蛋白的作用。这项工作与对白内障病因学和预防感兴趣的视觉研究人员有关，白内障是全球导致人类失明的主要原因。