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

描述(申请人提供)：GFP重建(跨突触伙伴的GFP)最初在无脊椎动物中开发，是一种基于基因可控的荧光系统，用于识别两个细胞(或细胞群体)之间的接触位置。它依赖于膜系分裂(即非功能性)GFP模块的表达，这些模块必须聚集在一起以重建GFP荧光(spGFP 1-10和spGFP 11)；由于这种功能互补需要膜之间的紧密结合(&lt；100 nm)，GRAP是识别两个细胞之间突触的强大系统。这项应用的目的是开发和验证用于哺乳动物和果蝇神经回路的GRAP方法。我们将开发一系列GRAP工具，包括靶向表达spGFP的病毒载体，在CRE或四环素诱导系统的控制下生产表达spGFP的转基因(和敲入)系，并在正交表达控制系统下设计含有GRAP组分的果蝇染色体。这些试剂将为GRAP表达提供时间和空间的选择性，并为电路图谱提供一个通用的平台。我们建议在两个不同的模型中测试这些组件：苍蝇的热敏电路屏幕和小鼠的Cre报告屏幕。我们还将设计一种多色抓取，作为一种新颖的“突触指纹”技术。我们期望这些研究的结果将大大增加可用于电路映射的工具库，并有助于描述正常和疾病的神经系统。

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.