Moffat Jason, Grueneberg Dorre A, Yang Xiaoping, Kim So Young, Kloepfer Angela M, Hinkle Gregory, Piqani Bruno, Eisenhaure Thomas M, Luo Biao, Grenier Jennifer K, Carpenter Anne E, Foo Shi Yin, Stewart Sheila A, Stockwell Brent R, Hacohen Nir, Hahn William C, Lander Eric S, Sabatini David M, Root David E
Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA.
Cell. 2006 Mar 24;124(6):1283-98. doi: 10.1016/j.cell.2006.01.040.
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 approximately 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.
为了在包括原代细胞和非分裂细胞在内的多种哺乳动物细胞类型中进行阵列式或混合式功能缺失筛选，我们正在开发针对人类和小鼠基因组的慢病毒短发夹RNA（shRNA）文库。该文库目前包含104,000个载体，用多个经过序列验证的构建体靶向22,000个人类和小鼠基因中的每一个。为了测试该文库用于阵列筛选的效用，我们开发了一种基于高内涵成像的筛选方法，以鉴定人类癌细胞有丝分裂进程所需的基因，并将其应用于一组针对1,028个人类基因的5,000种独特的表达shRNA的慢病毒阵列。该筛选鉴定出了几种已知的以及大约100种有丝分裂进程和增殖的候选调节因子；靶向同一基因的多个shRNA的可用性有助于对推定的命中结果进行功能验证。这项工作为功能缺失筛选提供了一种广泛适用的资源，以及将其应用于生物学发现的路线图。
