Synaptic-GFP Tags for Zebrafish

描述（由申请人提供）：GFP-标签在调节模式生物如C.优雅 类似地，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中的突触发育。 然而，这样的融合仍然在很大程度上未被利用的斑马鱼，即使鱼是唯一适合于突触发育的正向遗传解剖，因为它的快速发展，透明的身体在幼虫发育，和易于遗传操作。 随着斑马鱼的基因组序列接近完成，在斑马鱼中开发这样的工具已经成熟。 作为第一步，在斑马鱼开发一个研究计划，旨在解剖的分子机制，调节突触靶向选择的特异性，我建议创建一组GFP标记的突触蛋白，以促进在斑马鱼胚胎和幼虫体内突触形成的分析。 使用现代重组DNA技术（GAL 4/ DAS系统，网关克隆技术，转座子载体和细菌人工染色体）的组合，我建议创建载体表达斑马鱼突触蛋白-GFP融合和转基因斑马鱼动物表达这些突触蛋白-GFP构建体。 我们将集中讨论我们在C中展示的“移动”构造。elegans的强大和精确的表达和标记突触囊泡群体或突触的活性区域。 具体来说，我们最近创造了一种新的融合到C。线虫突触囊泡蛋白，可以使活体蠕虫的突触检测灵敏度提高10倍。 此外，该C.当在果蝇中表达时，线虫蛋白融合体强烈定位于神经肌肉接头，因此线虫融合体在物种间起作用。 初步研究表明，当在瞬时表达系统中的神经元中表达时，等效的斑马鱼融合形成斑点，表明其定位于突触。 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C。当类似的斑马鱼蛋白融合体在斑马鱼神经元中表达时，elegans活性区标签将允许斑马鱼活性区的可视化。

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.