Synaptic-GFP Tags for Zebrafish

斑马鱼突触 GFP 标签

基本信息

批准号：
7236193
负责人：
MICHAEL L NONET
金额：
$ 7.4万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-07-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7236193
关键词：
Animal Model Animals Antibodies Bacterial Artificial Chromosomes Caenorhabditis elegans Cells Chimeric Proteins Chromosome Pairing Cloning Communities Condition Cyclophosphamide/Fluorouracil/Prednisone Data Dendrites Destinations Detection Development Dissection Drosophila genus Embryo Event Fish Proteins Fishes Foundations Genetic Genetic Engineering Green Fluorescent Proteins Hippocampus (Brain)Human Imagery Label Larva Learning Life Localized Mediating Molecular Nervous system structure Neuromuscular Junction Neurons Organism Pathway interactions Play Population Pre-study Process Proteins Recombinants Research Research Personnel Role Signal Transduction Specificity Synapses Synaptic Vesicles System Techniques Technology Testing Transgenes Transgenic Animals Transgenic Organisms Work Zebrafish Zebrafish Proteins base cell type experience expression vector genetic manipulation genome sequencing in vivo intercellular communication member mutant nervous system development neurotrophic factor novel presynaptic programs promoter response synaptogenesis tool vector zebrafish genome

项目摘要

DESCRIPTION (provided by applicant): GFP-tags have been extremely useful in the genetic dissection of molecular pathways that regulate the formation of synapses in model organisms such as C. elegans. Similarly, many such fusion proteins are now being used to examine synaptic development in cultured mammalian neurons. However, such fusions remain largely unutilized in Zebrafish even though the fish is uniquely suited for forward genetic dissection of synaptic development because of its fast development, transparent body during larval development, and the ease of genetic manipulation. With the genome sequence of Zebrafish nearing completion, such tools are ripe for development in the Zebrafish. As a first step in developing a research program in Zebrafish aimed at dissecting the molecular mechanisms that regulate the specificity of synapse target selection, I propose to create a set of GFP-tagged synaptic proteins to facilitate the analysis of synapse formation in vivo in Zebrafish embryos and larvae. Using a combination of modern recombinant DNA technologies (the GAL4/ DAS system, Gateway cloning technology, transposon vectors, and bacterial artificial chromosomes), I propose to create vectors for expression of Zebrafish synaptic protein-GFP fusions and transgenic Zebrafish animals expressing these synaptic protein-GFP constructs. We will concentrate on 'moving' constructs that we have shown in C. elegans to express robustly and precisely and label either synaptic vesicle populations or the active zone domain of the synapse. Specifically, we have recently created a novel fusion to a C. elegans synaptic vesicle protein that allows for 10-fold more sensitive detection of synapses in live worms. Furthermore, this C. elegans fusion works across species as the worm protein fusion localizes robustly to the neuromuscular junction when expressed in Drosophila. Preliminary studies indicate that the equivalent Zebrafish fusion forms puncta when expressed in neurons in a transient expression system suggesting it localizes to synapses. We propose to develop this fusion in Zebrafish to label presynaptic specializations. Similarly, we propose to test whether several recently developed C. elegans active zone tags will permit the visualization of Zebrafish active zones when the analogous Zebrafish protein fusions are expressed in Zebrafish neurons.

描述（由申请人提供）：GFP 标签在调节模型生物（如秀丽隐杆线虫）突触形成的分子途径的遗传解析中非常有用。同样，许多此类融合蛋白现在被用于检查培养的哺乳动物神经元的突触发育。然而，这种融合在斑马鱼中仍未得到充分利用，尽管斑马鱼因其快速发育、幼体发育期间透明的身体以及易于基因操作而特别适合突触发育的正向遗传解剖。随着斑马鱼基因组序列的接近完成，在斑马鱼中开发此类工具的时机已经成熟。作为开发斑马鱼研究计划的第一步，旨在剖析调节突触目标选择特异性的分子机制，我建议创建一组 GFP 标记的突触蛋白，以促进斑马鱼胚胎体内突触形成的分析和幼虫。结合现代重组DNA技术（GAL4/DAS系统、Gateway克隆技术、转座子载体和细菌人工染色体），我建议创建用于表达斑马鱼突触蛋白-GFP融合体的载体以及表达这些突触蛋白的转基因斑马鱼动物-GFP构建体。我们将专注于我们在秀丽隐杆线虫中展示的“移动”结构，以稳健而精确地表达并标记突触小泡群体或突触的活动区域。具体来说，我们最近创建了一种与秀丽隐杆线虫突触小泡蛋白的新型融合蛋白，可以将活体蠕虫突触的检测灵敏度提高 10 倍。此外，这种秀丽隐杆线虫融合在跨物种中发挥作用，因为当在果蝇中表达时，蠕虫蛋白融合牢固地定位于神经肌肉接头。初步研究表明，当在瞬时表达系统中的神经元中表达时，等效的斑马鱼融合物会形成斑点，表明它定位于突触。我们建议在斑马鱼中开发这种融合来标记突触前特化。同样，我们建议测试几个最近开发的秀丽隐杆线虫活性区标签是否允许在斑马鱼神经元中表达类似的斑马鱼蛋白融合时可视化斑马鱼活性区。