GFP Reconstitution Across Synaptic Partners (GRASP) Methods to Dissect Mammalian

描述（由申请人提供）：GRASP (GFP Reconstitution Across Synaptic Partners)，最初在无脊椎动物中开发，是一种遗传可控的基于荧光的系统，用于识别两个细胞（或细胞群）之间的接触位点。它依赖于膜系裂（即非功能性）GFP模块的表达，这些模块必须聚集在一起才能重建GFP荧光（spGFP1-10和spGFP11）；由于这种功能性互补需要膜之间的紧密结合（<100 nm），因此GRASP是识别两个细胞之间突触的强大系统。本申请的目的是开发和验证用于哺乳动物和果蝇神经回路的GRASP方法。我们将生成一系列的GRASP工具，包括靶向表达spGFPs的病毒载体，在Cre或四环素诱导系统的控制下产生表达spGFPs的转基因（和敲入）系，并在正交表达控制系统下设计含有GRASP成分的果蝇染色体。总之，这些试剂将提供时间和空间选择性的把握表达，并提供一个通用的平台，电路绘图。我们建议在两种不同的模型中测试这些组件：果蝇的热感电路屏幕和小鼠的Cre报告屏幕。我们还将设计一种多色的GRASP作为一种新颖的“突触指纹”技术。我们期望这些研究的结果能够显著增强可用的电路测绘工具库，并有助于表征正常和病变的神经系统。

DESCRIPTION (provided by applicant): GRASP (GFP Reconstitution Across Synaptic Partners), originally developed in invertebrates, is a genetically controllable fluorescence-based system for identifying sites of contact between two cells (or cell populations). It relies on the expression of membrane-tethered split (i.e. non-functional) GFP modules that must come together to reconstitute GFP fluorescence (spGFP1-10 & spGFP11); since this functional complementation requires close apposition between membranes (<100 nm), GRASP is a powerful system to identify synapses between two cells. The objective of this application is to develop and validate GRASP methods for use in mammalian and Drosophila neural circuits. We will generate a battery of GRASP tools, including viral vectors for targeted expression of spGFPs, produce transgenic (and knock-in) lines expressing spGFPs under the control of the Cre or tetracycline inducible systems, and engineer Drosophila chromosomes harboring GRASP components under orthogonal expression control systems. Together, these reagents will afford temporal and spatial selectivity of GRASP expression, and provide a versatile platform for circuit mapping. We propose to test these components in two different models: a thermosensory circuit screen in flies and a Cre reporter screen in mice. We will also engineer a multicolor GRASP as a novel "synaptic fingerprinting" technique. We expect the results of these studies to significantly enhance the arsenal of tools available for circuit mapping, and be helpful in the characterization of normal and diseased nervous systems.