Using zebrafish as a model system for investigating lens development, aging and cataract.

使用斑马鱼作为模型系统来研究晶状体发育、衰老和白内障。

• 批准号: 10046498
• 负责人: Mason Posner
• 金额: $ 31.12万
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046498
• 关键词: Affect; Age; Aging; Alleles; Anatomy; Area; Biological Models; Biology; Blindness; CRISPR/Cas technology; Cataract; Cell Differentiation process; Crystalline Lens; Crystallins; DNA Binding; Data; Data Set; Disease; Dissection; Embryo; Epithelial; Epithelial Cells; Epithelium; Etiology; Fishes; Funding; Genes; Genetic; Genetic Transcription; Genome; Goals; Heat shock proteins; Heat-Shock Response; Homeostasis; Human; Individual; Knock-out; Laboratories; Lens Diseases; Lens Opacities; Lens development; Maintenance; Mammals; Methods; Modeling; Molecular Biology Techniques; Monitor; Mus; Mutate; Orthologous Gene; Phenotype; Physiology; Play; Post-Translational Protein Processing; Protein Family; Proteins; Proteomics; Publishing; Regulation; Regulator Genes; Research; Research Personnel; Role; Students; System; Techniques; Testing; Tissue Differentiation; Tissues; Universities; Work; Zebrafish; age related; alpha-Crystallins; base; comparative; cost efficient; differential expression; experience; experimental study; fiber cell; innovation; interest; lens; lens transparency; loss of function; member; mutant; novel; paralogous gene; prevent; promoter; protein aggregation; protein expression; success; therapy development; training opportunity; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; undergraduate student; university student

The goal of this proposal is to use zebrafish as a model system to identify novel regulators of ocular lens development and examine how loss of single and combined α-crystallins, small heat shock proteins important for lens function, affect lens aging and the regulation of lens crystallin expression. These experiments will produce a better understanding of lens development and maintenance of lens transparency important for developing treatments to prevent lens opacities, the leading cause of blindness worldwide. Work in our laboratory has helped make the zebrafish a powerful model system for studying lens development, function and disease. Undergraduate students supported by past funding have cloned and characterized three zebrafish α-crystallins and used CRISPR/Cas9 gene editing to disable each to examine the impact of their loss on early lens development. Here, we propose in Aim I to use our already produced knockout lines to model the impact of α-crystallin loss on the maintenance of lens transparency and the gene regulatory networks that control lens protein expression. This aim will include an integration of transcriptomic and proteomic analyses, along with anatomical examination of resulting lens phenotypes. We hypothesize that the resulting lens phenotypes will depend on the specific α-crystallin lost, providing a novel examination of αB-crystallin function due to the presence of two orthologs in zebrafish for the single-copy mammalian gene. In Aim II we will use transcriptomic data sets generated by our collaborators from mouse and zebrafish tissues to identify possible novel transcription factors (TFs) regulating lens development. We will then efficiently target these TFs using CRISPR/Cas9 methods that will allow us to screen for lens phenotypes directly in injected embryos. Mutant TF alleles that disrupt normal lens development will be carried into stable lines to detail the role they play in establishing and maintaining lens transparency. We expect that these two aims will efficiently identify novel regulators of lens development and clarify the role of α- crystallins in maintaining lens transparency. The experiments proposed will provide excellent training opportunities for undergraduates in a research setting with a strong track record of student success.

该提案的目标是使用斑马鱼作为模型系统，以确定新的调节剂， 眼透镜发育并检查单个和组合的α-晶状体蛋白、小热量的损失 休克蛋白对透镜功能很重要，影响透镜老化和透镜晶体蛋白的调节 表情这些实验将有助于更好地理解透镜的发展， 保持透镜的透明度对于开发防止透镜混浊的处理是重要的， 全球失明的主要原因。我们实验室的工作有助于使斑马鱼成为 研究透镜发育、功能和疾病的强大模型系统。本科 在过去的资助下，学生们克隆并描述了三种斑马鱼α-晶体蛋白 并使用CRISPR/Cas9基因编辑来禁用每一个，以检查它们的损失对早期癌症的影响。 透镜显影。在这里，我们建议在目标I中使用我们已经生产的敲除线来建模 α-晶状体蛋白缺失对透镜透明性的影响及其基因调控 控制透镜蛋白表达的网络。这一目标将包括整合转录组学 和蛋白质组学分析，沿着对所得到的透镜表型的解剖学检查。我们 假设所得的透镜表型将取决于特定的α-晶状体蛋白损失， 提供了一种新的检查α B-晶状体蛋白的功能，由于存在两个直系同源物， 斑马鱼的单拷贝哺乳动物基因。在Aim II中，我们将使用转录组数据集 由我们的合作者从小鼠和斑马鱼组织中产生， 调节透镜发育的转录因子（TF）。然后我们将有效地瞄准这些TF 使用CRISPR/Cas9方法，这将使我们能够直接在注射的晶状体中筛选透镜表型， 胚胎破坏正常透镜发育的突变TF等位基因将被携带到稳定的品系中， 详细说明了它们在建立和保持透镜透明度方面的作用。我们预计这些 两个目标将有效地确定新的调节透镜发展和澄清的作用， 晶状体蛋白在保持透镜透明度方面的作用。提出的实验将提供出色的 为本科生提供在研究环境中进行培训的机会， 成功