Tissue-specific labeling of endogenous proteins with split fluorescent proteins

使用分裂荧光蛋白对内源蛋白进行组织特异性标记

• 批准号: 10046696
• 负责人: Stephanie Woo
• 金额: $ 45.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046696
• 关键词: Address; Amino Acids; Animal Model; Biological Models; Biological Process; Biomedical Research; CRISPR/Cas technology; Cell Adhesion; Cells; Chimeric Proteins; Code; Complement; Complex; Cytoskeletal Proteins; Cytoskeleton; Data; Development; Embryo; Embryonic Development; Engineering; Epitopes; Fishes; Fluorescence; Fluorescence Microscopy; Gene Transfer Techniques; Genes; Genomics; Green Fluorescent Proteins; Guide RNA; In Vitro; Injections; Knock-in; Label; Length; Libraries; Messenger RNA; Methods; Molecular; Morphogenesis; Morphologic artifacts; Oligonucleotides; Organ; Pattern; Plasmids; Property; Protein Fragment; Proteins; Regulation; Regulator Genes; Regulatory Element; Research Personnel; Risk; Role; Running; Scientist; Shapes; Signal Transduction; Students; System; Techniques; Testing; Tissues; Transgenes; Transgenic Organisms; Work; Zebrafish; base; beta barrel; biological research; cell behavior; cell motility; cell type; design; experimental study; genomic locus; in vivo; interest; novel; overexpression; preservation; promoter; protein complex; protein expression; protein protein interaction; reconstitution; repaired; tool; undergraduate student

PROJECT SUMMARY Protein labeling by fusion with genetically encoded fluorescent proteins has been a powerful tool for investigating biological processes, allowing scientists to observe and analyze protein expression, localization, and dynamics in living cells. However, traditional approaches for expressing fluorescent fusion proteins possess drawbacks including potential overexpression artifacts, and new methods are needed, especially for in vivo studies. Our lab studies vertebrate organ formation using the zebrafish (Danio rerio) model system. In zebrafish and many other model organisms, expression of fluorescent fusion proteins is often achieved by injection of in vitro transcribed mRNA, which provides ubiquitous expression, or by transgenesis, which utilizes gene regulatory elements to drive spatially and/or temporally restricted expression. However, both approaches run the risk of producing overexpression artifacts. An alternative approach is to “knock-in” fluorescent coding sequences into the genetic locus for the protein of interest. Although this approach preserves endogenous regulation of expression, targeted insertion can be technically difficult to achieve. Moreover, many proteins are expressed quite broadly, and fluorescent protein tagging at the endogenous locus does not allow one to study the tissue- specific roles of such proteins. To overcome these limitations, we propose using a split fluorescent protein approach to achieve tissue-specific and endogenous protein labeling. Split fluorescent proteins consist of protein fragments that are expressed independently and possess little to no fluorescence on their own. However, when present in the same cell, the fragments self-assemble into a fluorescent complex. In this proposal, we will use a recently developed two- component system based on the green fluorescent protein mNeonGreen2 (split-NG). By expressing one component of the split-NG pair under a tissue specific promoter while fusing the second component to a protein of interest via genomic “knock-in”, our technique will enable tissue-specific examination of broadly expressed proteins. This technique has the potential to open new lines of inquiry in many fields of biological and biomedical research.

项目概要 通过与基因编码的荧光蛋白融合进行蛋白质标记已成为一种强大的工具 研究生物过程，使科学家能够观察和分析蛋白质表达、定位、 和活细胞的动力学。然而，表达荧光融合蛋白的传统方法 具有包括潜在的过度表达伪影在内的缺点，并且需要新的方法，特别是对于 体内研究。 我们的实验室使用斑马鱼（斑马鱼）模型系统研究脊椎动物器官的形成。在斑马鱼和 许多其他模型生物体，荧光融合蛋白的表达通常是通过注射体外来实现的 转录的 mRNA，提供无处不在的表达，或通过转基因，利用基因调控 驱动空间和/或时间限制表达的元件。然而，这两种方法都存在风险 产生过度表达伪影。另一种方法是将荧光编码序列“敲入” 感兴趣的蛋白质的遗传位点。尽管这种方法保留了内源性调节 表达式，定向插入在技术上很难实现。此外，还表达了许多蛋白质 从广义上讲，内源基因座上的荧光蛋白标记不允许人们研究组织- 此类蛋白质的特定作用。 为了克服这些限制，我们建议使用分裂荧光蛋白方法来实现组织特异性 和内源性蛋白质标记。分裂荧光蛋白由表达的蛋白质片段组成 独立且自身几乎不具有荧光。然而，当存在于同一细胞中时， 片段自组装成荧光复合物。在本提案中，我们将使用最近开发的两个- 基于绿色荧光蛋白 mNeonGreen2 (split-NG) 的成分系统。通过表达一 分裂NG对的一个组件在组织特异性启动子下，同时将第二个组件融合到 通过基因组“敲入”检测感兴趣的蛋白质，我们的技术将使广泛的组织特异性检查成为可能 表达的蛋白质。这项技术有可能在许多生物学领域开辟新的研究领域 和生物医学研究。