Tissue-specific labeling of endogenous proteins with split fluorescent proteins

项目摘要 通过与遗传编码的荧光蛋白融合的蛋白质标记已经成为一种强有力的工具， 研究生物过程，使科学家能够观察和分析蛋白质表达，定位， 以及活细胞的动力学。然而，表达荧光融合蛋白的传统方法 具有包括潜在的过表达伪像的缺点，并且需要新的方法，特别是对于在 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。在斑马鱼和 在许多其他模式生物中，荧光融合蛋白的表达通常通过体外注射 转录的mRNA，其提供普遍存在的表达，或通过转基因，其利用基因调控 元素来驱动空间和/或时间上受限的表达。然而，这两种方法都有风险， 产生过表达假象。另一种方法是将荧光编码序列“敲入”到 目标蛋白质的遗传位点。虽然这种方法保留了内源性调节， 表达，靶向插入在技术上可能难以实现。此外，许多蛋白质表达 广泛地说，荧光蛋白标记在内源基因座上不允许研究组织- 这些蛋白质的特殊作用。 为了克服这些局限性，我们建议使用分裂荧光蛋白的方法，以实现组织特异性 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， 并且它们自身具有很少或没有荧光。然而，当存在于同一细胞中时， 片段自组装成荧光复合物。在本建议中，我们将使用最近开发的两个- 基于绿色荧光蛋白mNeonGreen 2（split-NG）的组分系统。通过表达一个 在组织特异性启动子下融合分裂-NG对的第二组分，同时将第二组分融合至 通过基因组“敲入”，我们的技术将能够对广泛的组织特异性检查 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方向 和生物医学研究。

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.