Identification of GFP gene traps in zebrafish

斑马鱼 GFP 基因陷阱的鉴定

• 批准号: 6987039
• 负责人: STEPHEN L JOHNSON
• 金额: $ 31.37万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-15 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6987039
• 关键词: chimeric proteins; embryogenesis; fluorescent dye /probe; gene expression; gene mutation; genetic promoter element; genetic screening; genetically modified animals; green fluorescent proteins; ionophores; method development; nonmammalian vertebrate embryology; polymerase chain reaction; transposon /insertion element; zebrafish

DESCRIPTION (provided by applicant): The optical clarity of the zebrafish embryo, together with external development and the availability of genetic mutations, provides an ideal tool for analysis of embryogenesis and organogenesis. Full utilization of the zebrafish requires the availability of markers for individual cells or tissues that can be visualized in the living embryo. We will take advantage of efficient transposition of gene/enhancer traps engineered in the medaka tol2 transposon to identify lines expressing GFP in specific cells, tissues or organs. (1) We will identify upwards of 500 gene traps or enhancer traps strongly expressing GFP in specific cells, tissues or organs, in embryonic, larval, or adult stages. These GFP lines will be provided to individual researchers as they are isolated, archived as sperm or live stocks in the PI's facility, and submitted to the Zebrafish International Resource Center. (2) We will identify integration sites of isolated gene- or enhancer-trap lines, identify tagged genes, and determine whether the integration results in a visible or lethal mutation. (3) We will develop techniques for more efficient identification of gene or enhancer traps. We will test whether expression patterns in chimeric F0 injected animals reliably predicts patterns in F1s, or whether injection into eggs prior to fertilization will result in integration prior to the first mitotic DMA synthesis, resulting in non-mosaic expression. This work will enhance the use of zebrafish in developmental biology. It will identify live fluorescent markers for many of the cells, tissues and organs of the zebrafish embryo and larvae, identify the expression patterns for up to 500 tagged genes or enhancers, and generate recessive-lethal mutations in approximately 250 genes. This will enhance analysis of existing mutations, identify new molecularly identified mutations, and identify new phenomena in development of the vertebrate organism.

描述（由申请人提供）：斑马鱼胚胎的光学透明度以及外部发育和基因突变的可用性为分析胚胎发生和器官发生提供了理想的工具。斑马鱼的充分利用需要有可在活胚胎中可视化的单个细胞或组织的标记。我们将利用青鳉 tol2 转座子中基因/增强子陷阱的有效转座来鉴定在特定细胞、组织或器官中表达 GFP 的细胞系。 (1) 我们将在胚胎、幼虫或成虫阶段的特定细胞、组织或器官中鉴定出超过 500 个强表达 GFP 的基因陷阱或增强子陷阱。这些 GFP 系将在分离后提供给个别研究人员，作为精子或牲畜储存在 PI 设施中，并提交给斑马鱼国际资源中心。 (2) 我们将识别分离的基因或增强子捕获系的整合位点，识别标记基因，并确定整合是否导致可见突变或致死突变。 (3)我们将开发更有效地识别基因或增强子陷阱的技术。我们将测试注射嵌合 F0 的动物中的表达模式是否能可靠地预测 F1 中的模式，或者在受精前注射到卵中是否会导致第一次有丝分裂 DMA 合成之前的整合，从而导致非嵌合表达。 这项工作将增强斑马鱼在发育生物学中的应用。它将识别斑马鱼胚胎和幼虫的许多细胞、组织和器官的活荧光标记，识别多达 500 个标记基因或增强子的表达模式，并在大约 250 个基因中产生隐性致死突变。这将加强对现有突变的分析，识别新的分子识别突变，并识别脊椎动物发育中的新现象。