Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells
Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells
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DOI：
10.1073/pnas.1307002110
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发表时间：
2013-06-18
影响因子：
11.1
通讯作者：
Weissman, Jonathan S.
中科院分区：
文献类型：
--
作者：
Kampmann, Martin;Bassik, Michael C.;Weissman, Jonathan S.
后基因组时代的一个主要挑战是了解人类基因在正常和疾病状态下如何共同发挥作用。在微生物中，高密度遗传相互作用(GI)图谱是阐明基因功能和途径的有力工具。我们开发了一种基于哺乳动物细胞中汇集的shRNA筛选的综合方法学，用于全基因组范围内识别具有相关表型的基因，并系统地定位其中的所有GI。我们最近展示了这种方法在控制人类细胞对蓖麻毒素敏感性的途径中的应用潜力。在这里，我们提出了支持我们的策略的完整的定量框架，包括实验设计，从集合筛选中获得定量表型，使用超复杂shRNA文库稳健地识别HIT基因，并行测量来自单个双shRNA实验的数万个GI，以及构建GI图谱。我们描述了我们的策略的一般适用性。我们的混合方法可以在不同的细胞系和不同的条件下快速筛选相同的shRNA文库，以确定一系列不同的表型。我们在这里为单shRNA文库和双shRNA文库说明了这一策略。我们比较了不同的人类细胞系对蓖麻毒素和志贺毒素易感性基因的作用，揭示了毒素特异性和细胞系特异性的途径。我们还提供了基于生长和蓖麻毒素抗性表型的GI图谱，并展示了这种比较的GI图谱策略如何实现对物理复合体和上下文相关途径的功能剖析。
A major challenge of the postgenomic era is to understand how human genes function together in normal and disease states. In microorganisms, high-density genetic interaction (GI) maps are a powerful tool to elucidate gene functions and pathways. We have developed an integrated methodology based on pooled shRNA screening in mammalian cells for genome-wide identification of genes with relevant phenotypes and systematic mapping of all GIs among them. We recently demonstrated the potential of this approach in an application to pathways controlling the susceptibility of human cells to the toxin ricin. Here we present the complete quantitative framework underlying our strategy, including experimental design, derivation of quantitative phenotypes from pooled screens, robust identification of hit genes using ultra-complex shRNA libraries, parallel measurement of tens of thousands of GIs from a single double-shRNA experiment, and construction of GI maps. We describe the general applicability of our strategy. Our pooled approach enables rapid screening of the same shRNA library in different cell lines and under different conditions to determine a range of different phenotypes. We illustrate this strategy here for single-and double-shRNA libraries. We compare the roles of genes for susceptibility to ricin and Shiga toxin in different human cell lines and reveal both toxin-specific and cell line-specific pathways. We also present GI maps based on growth and ricin-resistance phenotypes, and we demonstrate how such a comparative GI mapping strategy enables functional dissection of physical complexes and context-dependent pathways.