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首页> 外文期刊>Current Protocols in Cell Biology >Fluorescence Activated Cell Sorting (FACS) in Genome-Wide Genetic Screening of Membrane Trafficking
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Fluorescence Activated Cell Sorting (FACS) in Genome-Wide Genetic Screening of Membrane Trafficking

机译：荧光活性细胞分选（FACS）在膜贩运基因组型遗传筛选中

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摘要

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 oflive 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.
机译：关于真核细胞中的细胞蛋白质的三分之一定位于在内膜系统膜包围的细胞器。细胞器中这些膜蛋白（包括可溶性内腔蛋白）的投放是通过囊样小泡介导的。囊泡介导的膜运输调节范围广泛的生物学过程，其中许多在分子水平仍然知之甚少。一个强大的方法来剖析一个小泡介导膜运输途径是公正的全基因组基因筛查，最近才在哺乳动物细胞成为可能用单倍体的人类细胞系的分离和CRISPR-Cas9基因组编辑的发展。在这里，我们描述了一种基于FACS的方法来选择群oflive基于内源性蛋白或改造的记者的表面水平的突变体细胞。这些突变群体的集合使得后续的深度测序和生物信息学分析，以确定调节运输途径的基因。这种方法可以容易地适合于遗传解剖广泛使用单倍体遗传学或CRISPR-Cas9屏幕哺乳动物膜运输过程。

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