Mapping endogenous protein dynamics in living cells

项目总结/摘要 后基因组时代的一个核心挑战是全面表征约20，000个基因的细胞作用。 人类基因组中编码的蛋白质。功能标签是一种强有力的策略，以表征细胞 蛋白质的作用。特别地，标签允许访问蛋白质功能的两个关键特征：定位（使用 荧光标签）和相互作用配偶体（使用表位标签和免疫沉淀）。因此，通过标记 蛋白质，一个生物体的蛋白质组的全面功能描述， 办妥了一批为此，我们先前开发了基于自互补分裂的FP 11标签 荧光蛋白，其与使用Cas9/sgRNA核糖核蛋白（RNP）的基因编辑组合， 能够在哺乳动物细胞系中快速、有效和高度可扩展地标记内源蛋白。虽然我们的 结果为大规模产生内源性标记的人细胞系铺平了道路， 蛋白质组范围内的分析蛋白质定位和相互作用网络在一个天然的细胞环境。然而，在这方面， 对于大规模库的实际生成和分析，仍然需要考虑几个主要的技术限制。 解决：亮度，颜色可用性，以及低光漂白的实时成像平台。拟议 项目，我们计划设计改进的FP 11标签和新的分裂蛋白片段标签，以解决这些技术问题。 挑战我们还将通过开发一种新的选择性飞机来展示强大的应用程序 在一个实施方案中，使用照明显微镜（SPIM）系统来筛选内源性标记的文库。

PROJECT SUMMARY/ABSTRACT A central challenge of the post-genomic era is to comprehensively characterize the cellular role of the ~20,000 proteins encoded in the human genome. Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immuno-precipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism’s proteome can be achieved. For this purpose, we have previously developed FP11 tags based on self-complementing split fluorescent proteins, which, in combination with gene editing using Cas9/sgRNA ribonucleoprotein (RNPs), enable rapid, efficient and highly scalable tagging of endogenous proteins in mammalian cell lines. While our results have paved the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. However, for practical generation and analysis of large-scale libraries, several major technical limitations still need to be addressed: brightness, color availability, and live imaging platforms with low photobleaching. In the proposed project, we plan to engineer improved FP11 tags and new split protein fragment tags to address these technical challenges. We will also demonstrate the powerful applications by developing a new selective plane illumination microscopy (SPIM) system to screen an endogenously tagged library.