Development and application ofa novel genetic strategy for visualization offunctional neural pathways with WGA-GFP fusion transgene

利用 WGA-GFP 融合转基因可视化功能神经通路的新型遗传策略的开发和应用

基本信息

批准号：
12480243
负责人：
YOSHIHARA Yoshihiko
金额：
$ 10.37万
依托单位：
RIKEN
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Information transfer between neurons takes place at the synapse. The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. Thus, detailed knowledge of neuronal networks is essential for understanding the wide range of brain fhrlctions. We previously developed a powerfirl genetic strategy for visualization of specific neuronal pathways across a synapse by combining a neuroanatomical tracing method with transgenic and gene targeting technology. By introducing cDNA for a plant lectit, wheat germ agglutinin (WGA), as a transgene under the control of specific promoter elements, selective and ftinctional transsynaptic neural pathways could be visualized. To detect the WGA transgene product immunohistochemically with anti-WGA antibody, however, the animals must be sacrificed and the tedious and time-consuming procedures are needed. For more convenient and simple detection of transsynaptic reporter, we attempted to develop fusion transgenes consisting of WGA and various fluorescent proteins (green : GFP, cyan : CFP, yellow : YFP, red : DsRed). If such chimeric molecules could be efficiently transferred across synapses with bright fluorescence, it would become possible to visualize functional neural pathways in situ or ultimately in vivo and to combine this transsynaptic tracing technique with various physiological measurements. We made several ftision transgenes and found that these molecules show bright fluorescence in cultured neurons. We are now checking whether the fluorescent WGA can be transferred across a synapse or not.

神经元之间的信息传递发生在突触。通过特定的突触连接之间的各种神经元之间的接线模式是大脑用于信息处理的功能逻辑的基础。因此，对神经元网络的详细知识对于理解广泛的大脑Fhrlctions至关重要。我们以前开发了一种通过将神经解剖学方法与转基因和基因靶向技术相结合，用于通过突触中特定神经元途径可视化特定神经元途径的遗传策略。通过在特定启动子元素控制下作为转基因引入植物夹具的cDNA，小麦胚芽凝集素（WGA）作为转基因，可以看到选择性和ftinct性的跨性神经途径。为了用抗WGA抗体检测WGA转基因产物免疫组织化学，但是必须处死动物，并且需要进行乏味和耗时的程序。为了更方便，更简单地检测到透射记者，我们尝试开发由WGA和各种荧光蛋白组成的融合基因（绿色：GFP，CYAN：CFP，CFP，黄色：YFP，YFP，红色：DSRED：DSRED）。如果可以有效地在突触中以明亮的荧光进行有效传递这种嵌合分子，则可以看到原位或最终在体内的功能性神经途径，并将这种透射性跟踪技术与各种生理测量结合在一起。我们进行了几个Ftision转基因，发现这些分子在培养的神经元中显示出明亮的荧光。现在，我们正在检查是否可以将荧光WGA转移到突触中。