Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158, USA.
Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):E2317-26. doi: 10.1073/pnas.1307002110. Epub 2013 Jun 5.
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.
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。我们已经开发了一种基于哺乳动物细胞中 pooled shRNA 筛选的综合方法，用于在全基因组范围内鉴定具有相关表型的基因，并对它们之间的所有 GI 进行系统映射。我们最近在应用于控制人类细胞对毒素蓖麻毒素易感性的途径的应用中证明了这种方法的潜力。在这里，我们介绍了我们策略的完整定量框架，包括实验设计、从 pooled 筛选中得出定量表型、使用超复杂 shRNA 文库稳健识别命中基因、从单个双 shRNA 实验中平行测量数万条 GI 以及构建 GI 图谱。我们描述了我们策略的一般适用性。我们的 pooled 方法能够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。我们在这里展示了用于单 shRNA 文库和双 shRNA 文库的这种策略。我们比较了不同人类细胞系中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途径。我们还基于生长和蓖麻毒素抗性表型呈现了 GI 图谱，并展示了这种比较 GI 映射策略如何能够对物理复合物和上下文相关途径进行功能剖析。
