FLASH/REASH LABELING OF TETRACYSTEINE-TAGGED PROTEINS IN ZEBRAFISH

斑马鱼中四半胱氨酸标记蛋白的 Flash/ReASH 标记

• 批准号: 7601045
• 负责人: MANFRED AUER
• 金额: $ 1.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601045
• 关键词: Animals; Antibodies; Binding; Biological Preservation; Cell Line; Chimeric Proteins; Computer Retrieval of Information on Scientific Projects Database; Detection; Electron Microscopy; Embryo; Exposure to; Fertilization; Fluorescence; Fluorescence Microscopy; Freezing; Funding; Goals; Grant; Image; Institution; Label; Light; Localized; Mediating; Methods; Microinjections; Mitochondria; Modeling; Noise; Organ; Proteins; Research; Research Personnel; Resolution; Resources; Signal Transduction; Source; Testing; United States National Institutes of Health; Vertebrates; Viral; Zebrafish; cell type; day; electron tomography; fluorophore; interest; pressure; promoter; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to become familiar with the technical aspects of labeling zebrafish embryos that express proteins genetically altered to contain a tetracysteine tag. This tag is recognized by the commercially available fluorophores FlAsH-EDT2 and ReAsH-EDT2. Binding of either fluorophore to the tetracysteine tag results in a fluorescent signal that can photoconvert diaminobenzidine into an osmiophilic precipitate. In combination with electron tomography, this method will enable us to localize any protein of interest with sub-cellular resolution and without raising antibodies. The zebrafish is a particularly attractive model for labeling proteins with a genetically-encoded universal tag: First, zebrafish develop much faster than higher vertebrates; as a result, most organs and cell types can be observed within a few days after fertilization. Second, animals that transiently or stably express tagged proteins can be generated simply by microinjecting embryos. Third, zebrafish embryos are transparent and easily penetrated by light, a prerequisite for fluorescence detection and photoconversion. Finally, the small zebrafish embryos can be frozen at high pressure and freeze-substituted, resulting in faithful preservation for electron microscopy. To test the universal-tag approach in zebrafish, we first created an expression construct that encodes yellow fluorescent protein (YFP) with a mitochondrial targeting signal under the control of a strong viral promoter both in HEK cell lines and zebrafish. Pilot experiments have demonstrated targeting of the mito-YFP fusion protein to mitochondria and strong mosaic expression upon microinjection of this construct into zebrafish embryos. We are now adding an optimized tetracystein tag (FLNCCPGCCMEP) to the fusion protein. The fluorescence spectra of YFP and ReAsH do not overlap significatnly, as their excitation/emission maxima lie at 512/529 and 593/608 nm, respectively. At the NCMIR, we propose to examine the binding of the ReAsH fluorophore in microinjected zebrafish embryos to the mito-YFP-TetraCys protein or to the mito-YFP negative control. First, we will vary the ReAsH concentration and the duration of exposure to optimize the signal-to-noise ratio. If we observe specific binding to the mito-YFP-TetraCys protein, as determined by fluorescence microscopy, we will proceed to the ReAsH-mediated photoconversion of diaminobenzidin in order to perform TEM imaging.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该项目的目的是熟悉标记斑马鱼胚胎的技术方面，这些斑马鱼胚胎表达遗传上改变以包含四半半胱氨酸标签的蛋白质。该标签通过市售的荧光团闪存-EDT2和REASH-EDT2识别。两种荧光团与四环素状态标签的结合会导致荧光信号，该信号可以将二氨基苯胺光化为渗透性沉淀物。结合电子断层扫描，这种方法将使我们能够通过亚细胞分辨率定位任何感兴趣的蛋白质，而无需提高抗体。斑马鱼是一个特别有吸引力的模型，用于用遗传编码的通用标签标记蛋白质：首先，斑马鱼的发展速度比较高的脊椎动物快得多。结果，大多数器官和细胞类型都可以在受精后的几天内观察到。其次，可以简单地通过微注射胚胎产生瞬时或稳定表达标记蛋白的动物。第三，斑马鱼胚胎是透明的，很容易被光穿透，这是荧光检测和光转化的先决条件。最后，可以在高压和冷冻取代的小斑马鱼胚胎中冷冻，从而忠实地保存电子显微镜。为了测试斑马鱼中的通用标签方法，我们首先创建了一种表达构建体，该构建体在HEK细胞系和斑马鱼中编码具有线粒体靶向信号的黄色荧光蛋白（YFP）。试验实验表明，在将这种构建体显微注射到斑马鱼胚胎中后，将线粒体-YFP融合蛋白靶向线粒体和强镶嵌表达。现在，我们正在为融合蛋白添加优化的四环标签（FLNCCPGCCMEP）。 YFP和REASH的荧光光谱并不能显着重叠，因为它们的激发/发射最大值分别位于512/529和593/608 nm。在NCMIR，我们建议检查斑马鱼胚胎中荧光团与mito-YFP-Tetracys蛋白或Mito-YFP阴性对照的结合。首先，我们将改变重新浓度和暴露持续时间，以优化信噪比。如果我们观察到与荧光显微镜确定的特异性结合与Mito-YFP-Tetracys蛋白，我们将继续进行二氨基苯甲酸蛋白的reash介导的光转化，以执行TEM成像。