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Mapping endogenous protein dynamics in living cells

绘制活细胞内源蛋白质动态图

基本信息

批准号：
10473533
负责人：
Bo Huang
金额：
$ 28.05万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-20 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10473533
关键词：
3-Dimensional Address CRISPR/Cas technology Cell Cycle Cell Line Cell Nucleus Cells Cellular biology Collection Color Comb animal structure Complement Consumption DNA Development Directed Molecular Evolution Engineering Ensure Epitopes Gene Proteins Generations Genes Genomics Human Human Cell Line Human Genome Image Immunoprecipitation Knock-in Label Laboratories Libraries Lighting Mammalian Cell Maps Methods Microscopy Mitosis Nuclear Proteins Organism Performance Photobleaching Process Protein Analysis Protein Dynamics Protein Engineering Protein Fragment Proteins Proteome Resolution Resources Ribonucleoproteins Role Saccharomyces cerevisiae Saccharomycetales Sampling Signal Transduction System Time base design experience fluorescence imaging fluorescence microscope genome-wide imaging platform improved instrumentation live cell microscopy multidisciplinary protein function screening success tool yeast protein

项目摘要

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.

项目总结/文摘