Fluorescence labeling of PSD-95 at endogenous levels for single cell imaging

内源水平 PSD-95 的荧光标记用于单细胞成像

• 批准号: 8702775
• 负责人: Haining Zhong
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702775
• 关键词: AMPA Receptors; Address; Affect; Age; Animals; Brain; C-terminal; Cell physiology; Cells; Code; Communication; Complement; Cre-LoxP; DLG4 gene; Disease; Electroporation; Exons; Fluorescence; Functional disorder; Funding; Future; Gene Targeting; Genes; Genetic Recombination; Glutamates; Golgi Apparatus; Habitats; Human; Image; Imagery; Immunoelectron Microscopy; In Situ; Individual; Knock-in Mouse; Knock-out; Knowledge; Label; Life; Light; Mediating; Messenger RNA; Methods; Microscopy; Monitor; Mus; Names; Neurodegenerative Disorders; Neurons; Physiology; Polyadenylation; Population; Preparation; Property; Protein Dynamics; Proteins; Regulation; Resolution; Sampling; Shapes; Signal Transduction; Site; Staining method; Stains; Structure; Synapses; Synaptic plasticity; Techniques; Technology; Testing; Tissues; Transcriptional Regulation; Transgenic Mice; Translational Regulation; Vertebral column; Virus Diseases; brain tissue; cell type; cellular imaging; design; genetic manipulation; human disease; in utero; in vivo; innovation; interest; mouse model; nervous system disorder; novel; overexpression; postsynaptic; protein function; public health relevance; recombinase; spatiotemporal; stoichiometry; synaptic function; trafficking; two-photon

DESCRIPTION (provided by applicant): Brain function relies on the coordinated actions of trillions of tiny specialized structures called synapses, which serve as communication gateways between neurons. Small changes in these synaptic machineries are the early manifestation of many neurological disorders and neurodegenerative diseases, which become increasingly prevalent as the population ages. Proper synaptic function relies on the precise, yet dynamic, regulation of the abundance and subcellular localization of synaptic proteins. Monitoring the spatiotemporal organization of these proteins in live neurons under native conditions is an important step toward understanding their function. However, it remains challenging to visualize endogenous synaptic protein organization in neurons in their native habitat - in living brain tissue or in living animals. The majority of studies investigating protein dynamics rely on the overexpression of fluorescently tagged proteins of interest. Unfortunately, overexpression can alter protein stoichiometry, trafficking, subcellular localization, and signaling, ultimately affecing cellular function. Although 'knock-in' strategies can, in principle, be used to label the target molecule with a fluorescent protein and express it at endogenous levels, most knock-in approaches result in the global expression of the labeled protein. Global expression leads to high background fluorescence and a lack of cell-specific contrast in intact tissue, making high-resolution imaging studies of protein dynamics difficult. To address these problems we propose to develop an innovative mouse knock-in strategy called Conditional Labeling by Exon Duplication, or CLED, to fluorescently tag specific proteins and express them at endogenous levels in a sparse subset of neurons. By using an innovative approach that combines the Cre/LoxP site- specific recombination strategy with exon duplication, we will express the tagged proteins under the control of endogenous transcriptional and translational regulation while maintaining cell-specific, Golgi-staining-like high contrast. As a proof of principle, in Specific im 1, we will tag the critical synaptic protein PSD-95 with the yellow fluorescent protein mVenus, and, in Specific Aim 2, we will use the resulting transgenic mouse line to examine functionally significant dynamics of PSD-95 organization. Once established, our proposed strategy will complement current microscopy techniques and sample preparation methods to provide a previously unattainable, highly sensitive, dynamic view of the spatiotemporal organization of specific synaptic proteins in situ and in vivo. The same strategy can be applied to visualize proteins that carry out non-neuronal functions. The acquired knowledge and principles from these novel transgenic mouse lines will facilitate future studies of human brain function and disease.

描述（由申请人提供）：大脑功能依赖于数万亿称为突触的微小专门结构的协调动作，这些结构充当神经元之间的通信网关。这些突触机制的微小变化是许多神经系统疾病和神经退行性疾病的早期表现，随着人口老龄化，这些疾病变得越来越普遍。正确的突触功能依赖于突触蛋白的丰度和亚细胞定位的精确而动态的调节。在自然条件下监测这些蛋白质在活神经元中的时空组织是了解其功能的重要一步。然而，在活体脑组织或活体动物的自然栖息地中，可视化神经元中的内源性突触蛋白组织仍然具有挑战性。研究蛋白质动力学的大多数研究依赖于感兴趣的荧光标记蛋白质的过表达。不幸的是，过表达可以改变蛋白质的化学计量、运输、亚细胞定位和信号传导，最终影响细胞功能。虽然“敲入”策略原则上可以用于用荧光蛋白标记靶分子并在内源水平表达它，但大多数敲入方法导致标记蛋白的全局表达。全局表达导致高背景荧光和完整组织中细胞特异性对比度的缺乏，使得蛋白质动力学的高分辨率成像研究变得困难。为了解决这些问题，我们建议开发一种创新的小鼠敲入策略，称为外显子复制条件标记（CLED），以荧光标记特定蛋白质并在稀疏的神经元子集中以内源性水平表达它们。通过使用将Cre/LoxP位点特异性重组策略与外显子复制相结合的创新方法，我们将在内源性转录和翻译调控的控制下表达标记的蛋白质，同时保持细胞特异性的高尔基体染色样高对比度。作为原理的证明，在Specific im 1中，我们将用黄色荧光蛋白mVenus标记关键突触蛋白PSD-95，并且在Specific Aim 2中，我们将使用所得转基因小鼠系来检查PSD-95组织的功能显著动态。一旦建立，我们提出的策略将补充目前的显微镜技术和样品制备方法，提供一个以前无法实现的，高度敏感的，动态的特定突触蛋白在原位和体内的时空组织的看法。同样的策略可以应用于可视化执行非神经元功能的蛋白质。从这些新的转基因小鼠品系中获得的知识和原理将有助于未来对人脑功能和疾病的研究。