Pooled Lentiviral shRNA Screening for Functional Genomics in Mammalian Cells
Pooled Lentiviral shRNA Screening for Functional Genomics in Mammalian Cells
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DOI：
10.1007/978-1-61779-276-2_9
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Moffat, Jason
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文献类型：
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作者：
Blakely, Kim;Ketela, Troy;Moffat, Jason
基因组测序工作改变了生物学研究的性质，促进了哺乳动物基因功能注释技术的发展。基于最初在植物和秀丽隐杆线虫中发现的方法，RNA干扰为细胞生物学家提供了一种有效且可重复的方法来干扰哺乳动物细胞和整个生物体的基因功能。靶向人类和小鼠基因组的RNA干扰文库的最初应用依赖于阵列筛选方法，由此将每种独特的RNA干扰试剂排列到微量滴定板的各个威尔斯孔中。这些屏幕执行起来并不简单，需要对基础设施进行大量投资。在过去的十年中，已经取得了许多技术进步，使全基因组RNA干扰筛选更容易为研究人员所用，并且在非专业实验室中更可行。在这里，我们描述了一个全面的协议，汇集短发夹RNA筛选，包括方法汇集慢病毒生产，细胞感染，全基因组负选择筛选和资源汇集屏幕反卷积，和数据分析。作为一种技术，汇集的shRNA筛选仍处于起步阶段，但该方法已经成功地应用于探测不同的信号通路，作为药物靶点鉴定的手段，并在正常和癌细胞系中鉴定必需基因。
Genome sequencing efforts have reformed the nature of biological inquiry, prompting the development of technologies for the functional annotation of mammalian genes. Based on methodologies originally discovered in plants and Caenorhabditis elegans., RNA interference has offered cell biologists an effective and reproducible approach to perturb gene function in mammalian cells and whole organisms. Initial application of RNA interference libraries targeting the human and mouse genomes relied on arrayed screening approaches, whereby each unique RNA interference reagent is arrayed into individual wells of a microtiter plate. These screens are not trivial to perform, requiring a substantial investment in infrastructure. In the past decade, many technological advances have been made that make genome-wide RNA interference screening more accessible to researchers and more feasible to perform in nonspecialized laboratories. Here, we describe a comprehensive protocol for pooled short-hairpin RNA screening, including methodologies for pooled lentivirus production, cell infection, genome-wide negative selection screening and resources for pooled screen deconvolution, and data analysis. As a technique, pooled shRNA screening is still in its infancy, hut the methodology has already been successfully applied to probe diverse signaling pathways, as a means of drug target identification, and to identify essential genes in normal and cancer cell lines.