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

基本信息

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
项目类别：
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

项目摘要

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生物学研究过程，使科学家能够观察和分析蛋白质表达、定位、然而，表达荧光融合蛋白的传统方法。存在缺陷，包括潜在的过度表达伪影，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（Danio rerio）模型系统研究脊椎动物器官的形成。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，利用基因调控然而，这两种方法都存在以下风险：另一种方法是将荧光编码序列“敲入”。尽管这种方法保留了感兴趣蛋白质的遗传位点。表达，靶向插入在技术上可能难以实现，而且，许多蛋白质都被表达。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分裂荧光蛋白由表达的蛋白质片段组成。然而，当存在于同一细胞中时，它们本身不具有荧光。在本提案中，我们将使用最近开发的双片段片段自组装成荧光复合物。基于绿色荧光蛋白 mNeonGreen2 (split-NG) 的组分系统通过表达一种。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该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。