A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen
A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen
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DOI：
10.1016/j.cell.2006.01.040
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发表时间：
2006-03-24
期刊：
影响因子：
64.5
通讯作者：
Root, DE
中科院分区：
文献类型：
--
作者：
Moffat, J;Grueneberg, DA;Root, DE
为了在广泛的哺乳动物细胞类型，包括原代细胞和非分裂细胞中进行排列或汇集的功能丧失筛查，我们正在开发针对人类和小鼠基因组的慢病毒短发夹状RNA(ShRNA)文库。这些文库目前包含104,000个载体，针对22,000个人类和小鼠基因中的每一个，具有多个序列验证的构建体。为了测试该文库用于阵列筛查的实用性，我们开发了一种基于高含量成像的筛查，以确定人类癌细胞有丝分裂进展所需的基因，并将其应用于一组针对1,028个人类基因的5,000个独特的shRNA表达的慢病毒阵列。该筛选确定了几个已知的和类似的100个有丝分裂进展和增殖的候选调控因子；针对同一基因的多个shRNA的可用性促进了推测的HITS的功能验证。这项工作为功能丧失筛查提供了广泛适用的资源，并为其在生物发现中的应用提供了路线图。
To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and similar to 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.