Synaptic-GFP Tags for Zebrafish

描述（由申请人提供）：gfp标签在模式生物（如秀丽隐杆线虫）中调节突触形成的分子途径的遗传解剖中非常有用。同样，许多这样的融合蛋白现在被用来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，尽管斑马鱼由于其快速发育、幼虫发育期间身体透明以及易于遗传操作而特别适合于突触发育的前向遗传解剖。随着斑马鱼基因组序列的接近完成，这些工具在斑马鱼身上的发展已经成熟。作为在斑马鱼中开展研究计划的第一步，旨在解剖调节突触靶点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，以促进斑马鱼胚胎和幼虫体内突触形成的分析。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），我建议创建表达斑马鱼突触蛋白- gfp融合的载体和表达这些突触蛋白- gfp构建体的转基因斑马鱼动物。我们将专注于我们在秀丽隐杆线虫中展示的“移动”结构，以强大而精确地表达并标记突触囊泡种群或突触的活跃区域。具体来说，我们最近创造了一种新的与秀丽隐杆线虫突触囊泡蛋白的融合，这种融合使得对活蠕虫突触的检测灵敏度提高了10倍。此外，当线虫蛋白在果蝇中表达时，这种线虫蛋白融合在神经肌肉连接处强烈地定位，因此这种线虫融合在物种间也能起作用。初步研究表明，斑马鱼的等效融合在神经元中的瞬时表达系统中形成点，这表明它定位于突触。我们建议在斑马鱼中发展这种融合来标记突触前特化。同样，我们建议测试几种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显示斑马鱼的活性区。

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.