Menasche Bridget L, Crisman Lauren, Gulbranson Daniel R, Davis Eric M, Yu Haijia, Shen Jingshi
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado.
Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China.
Curr Protoc Cell Biol. 2019 Mar;82(1):e68. doi: 10.1002/cpcb.68. Epub 2018 Sep 28.
About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing. Here, we describe a FACS-based method to select populations of live mutant cells based on the surface levels of endogenous proteins or engineered reporters. Collection of these mutant populations enables subsequent deep sequencing and bioinformatics analysis to identify genes that regulate the trafficking pathway. This method can be readily adapted to genetically dissect a broad range of mammalian membrane trafficking processes using haploid genetics or CRISPR-Cas9 screens. © 2018 by John Wiley & Sons, Inc.
真核细胞中约三分之一的细胞蛋白定位于内膜系统中被膜包裹的细胞器。这些膜蛋白（包括可溶性腔蛋白）在细胞器之间的运输由小囊状囊泡介导。囊泡介导的膜运输调节广泛的生物学过程，其中许多在分子水平上仍知之甚少。剖析囊泡介导的膜运输途径的一个有力方法是无偏向的全基因组遗传筛选，直到最近随着单倍体人类细胞系的分离和CRISPR-Cas9基因组编辑技术的发展，这种筛选才在哺乳动物细胞中成为可能。在这里，我们描述了一种基于荧光激活细胞分选（FACS）的方法，用于根据内源性蛋白或工程报告基因的表面水平选择活的突变细胞群体。收集这些突变群体能够进行后续的深度测序和生物信息学分析，以鉴定调节运输途径的基因。这种方法可以很容易地应用于利用单倍体遗传学或CRISPR-Cas9筛选对广泛的哺乳动物膜运输过程进行遗传学剖析。© 2018年约翰威立父子公司版权所有
