Tissue-specific labeling of endogenous proteins with split fluorescent proteins

项目总结 通过与基因编码的荧光蛋白融合来标记蛋白质已经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的表达、定位、 以及活细胞中的动力学。然而，传统的表达荧光融合蛋白的方法 具有缺陷，包括潜在的过度表达伪影，需要新的方法，特别是在 活体研究。 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 许多其他模式生物，荧光融合蛋白的表达通常是通过在体外注射 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 元素来驱动空间和/或时间受限的表达。然而，这两种方法都存在以下风险 制造过度表达的人工制品。另一种方法是将“敲入”的荧光编码序列 目标蛋白质的遗传位点。尽管这种方法保留了对 表达时，定向插入在技术上可能很难实现。此外，还表达了许多蛋白质。 相当广泛地说，在内源性位置标记荧光蛋白不允许人们研究组织- 这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 和内源蛋白标记。分裂的荧光蛋白由表达的蛋白质片段组成 独立的，本身几乎没有荧光的。但是，当出现在同一单元格中时， 碎片自组装成一个荧光复合体。在这项提案中，我们将使用最近开发的两种- 基于绿色荧光蛋白mNeonGreen2(Split-NG)的组成系统。通过表达一个 在组织特异性启动子下的Split-NG对的成分，同时将第二成分融合到 通过基因组“敲入”感兴趣的蛋白质，我们的技术将使组织特异性检查广泛 表达的蛋白质。这项技术有可能在生物学的许多领域开辟新的研究路线 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.