Identification of GFP gene traps in zebrafish

描述(申请人提供)：斑马鱼胚胎的光学清晰度，加上外部发育和基因突变的可用性，为胚胎发生和器官发生分析提供了理想的工具。斑马鱼的充分利用需要能够在活胚胎中看到的单个细胞或组织的标记。我们将利用青竹tol2转座子中设计的基因/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的系。 (1)我们将在胚胎、幼虫或成体阶段的特定细胞、组织或器官中鉴定500多个强表达GFP的基因陷阱或增强子陷阱。这些GFP品系将在分离后提供给个别研究人员，作为精子或活体存放物保存在PI的设施中，并提交给斑马鱼国际资源中心。(2)我们将确定分离的基因或增强子陷阱系的整合位置，识别标记的基因，并确定整合是否导致可见或致命的突变。(3)我们将开发更有效地识别基因或增强子陷阱的技术。我们将测试嵌合F0注射动物中的表达模式是否可靠地预测F1中的模式，或者在受精前注射到卵子中是否会导致第一次有丝分裂DMA合成之前的整合，从而导致非镶嵌表达。这项工作将加强斑马鱼在发育生物学中的应用。它将为斑马鱼胚胎和幼虫的许多细胞、组织和器官识别活的荧光标记，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命突变。这将加强对现有突变的分析，识别新的分子识别突变，并识别脊椎动物有机体发育中的新现象。

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.