Transgenic Studies of Vertebrate Retinal Development

脊椎动物视网膜发育的转基因研究

基本信息

批准号：
7253199
负责人：
JOHN E DOWLING
金额：
$ 36.71万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7253199
关键词：
Affect Anatomy Biochemical Biological Breeding Cells Cellular biology Chemistry Collection Complex Defect Development Developmental Process Embryo Ethylnitrosourea Exhibits Exposure to Eye Abnormalities Eye Development Family Female Fertilization Fishes Fluorescence Microscopy Generations Genes Genetic Genetic Screening Goals Green Fluorescent Proteins Histology Hour Immunohistochemistry In Situ Hybridization Institutes Invasive Invertebrates Libraries Life Measures Methodology Methods Monitor Mutagenesis Mutation Neuraxis Nomarski Interference Contrast Microscopy Numbers Opsin Pathway interactions Pattern Phenotype Physiological Physiology Proteins Rest Retina Retinal Rhodopsin Screening procedure Signal Transduction Standards of Weights and Measures System Time Transgenes Transgenic Organisms Xenopus Zebrafish behavior test cell type chemical genetics day density fluorescence microscope interest light microscopy male mutant neuroblast novel optic cup promoter protein expression retinal neuron retinal rods retinogenesis small molecule small molecule libraries tool transgene expression

项目摘要

DESCRIPTION (provided by applicant): To examine in more detail the developmental processes in the vertebrate retina, new screening methods will be used in zebrafish. Specifically, transgenic zebrafish expressing the green fluorescent protein (GFP) under a rod photoreceptor specific promoter will be used in two ways: A) A forward genetic screen to identify recessive mutations affecting rod development. Homozygous male transgenic zebrafish will be mutagenized by exposure to ethylnitrosourea. Mutagenized zebrafish will be bred to homozygous transgenic females to produce F2 generation families that are homozygous for the transgene. To identify recessive mutations, F3 generation fish will be examined by light and fluorescence microscopy for changes in GFP expression, indicating an alteration in rod photoreceptor number and/or retinal differentiation. B) Chemical genetic screens to identify molecles that disrupt pathways involved in proper retinal development and rod differentiation. Transgenic embryos will be arrayed at a density of 3 embryos/well of a 96-well plate and exposed to small molecules from libraries of known bioactive substances. The small molecule libraries consist of 2489 compounds and were assembled to contain molecules with known biological activity and with previously identified protein targets. Embryos will be exposed to small molecules at three timepoints significant in eye development: 9 hours post fertilization (hpf), when optic cup formation begins; 24 hpf, a time of retinoblast proliferation but prior to cellular differentiation; 48 hpf, the onset of rod photoreceptor differentiation. Embryos will be examined by standard light microscopy to screen for general morphological defects, differential interference contrast (DIC) microscopy to screen for defects in retinal lamination, and fluorescence microscopy to screen for defects in rod photoreceptor differentiation. Upon identification of specific mutations or small molecules, the phenotypes will be characterized by histology, immunohistochemistry, in situ hybridization, and mosaic analysis, when necessary. The goal of these studies will be to identify pathways in retinal development that could be missed in routine forward genetic screens.

描述（由申请人提供）：为了更详细地检查脊椎动物视网膜的发育过程，将在斑马鱼中使用新的筛选方法。具体来说，在视杆光感受器特异性启动子下表达绿色荧光蛋白（GFP）的转基因斑马鱼将以两种方式使用：A）正向遗传筛选，以识别影响视杆发育的隐性突变。纯合雄性转基因斑马鱼会因暴露于乙基亚硝基脲而发生诱变。诱变斑马鱼将与纯合转基因雌性交配，产生转基因纯合的 F2 代家族。为了识别隐性突变，将通过光学和荧光显微镜检查 F3 代鱼 GFP 表达的变化，表明视杆光感受器数量和/或视网膜分化的变化。 B) 化学遗传筛选，以识别破坏视网膜正常发育和视杆细胞分化途径的分子。转基因胚胎将以 3 个胚胎/孔的密度排列在 96 孔板中，并暴露于来自已知生物活性物质文库的小分子。小分子库由 2489 种化合物组成，经过组装后包含具有已知生物活性和先前鉴定的蛋白质靶标的分子。胚胎将在对眼睛发育具有重要意义的三个时间点接触小分子：受精后 9 小时 (hpf)，此时视杯开始形成； 24 hpf，视网膜母细胞增殖的时间，但在细胞分化之前； 48 hpf，杆状光感受器分化开始。将通过标准光学显微镜检查胚胎以筛查一般形态缺陷，通过微分干涉对比（DIC）显微镜检查以筛查视网膜层压缺陷，并通过荧光显微镜检查以筛查视杆光感受器分化的缺陷。在鉴定出特定突变或小分子后，必要时将通过组织学、免疫组织化学、原位杂交和镶嵌分析来表征表型。这些研究的目标是确定常规正向遗传筛查中可能遗漏的视网膜发育途径。