Silva Jose M, Li Mamie Z, Chang Ken, Ge Wei, Golding Michael C, Rickles Richard J, Siolas Despina, Hu Guang, Paddison Patrick J, Schlabach Michael R, Sheth Nihar, Bradshaw Jeff, Burchard Julia, Kulkarni Amit, Cavet Guy, Sachidanandam Ravi, McCombie W Richard, Cleary Michele A, Elledge Stephen J, Hannon Gregory J
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Nat Genet. 2005 Nov;37(11):1281-8. doi: 10.1038/ng1650. Epub 2005 Oct 2.
Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.
功能丧失型表型往往是理解生化途径的联系和生物学功能的关键。我们和其他研究团队之前构建了短发夹RNA文库，可用于系统分析RNA干扰在哺乳动物细胞中诱导的表型。在此，我们报告利用对RNA干扰生物化学的更多了解设计的第二代短发夹RNA表达文库的构建和验证。这些构建体包括设计用于模拟天然微小RNA初级转录本的沉默触发子，并且每个靶序列都是基于热力学标准选择的，以实现最佳的小RNA性能。生化和表型分析表明，新文库相比第一代试剂有显著改进。我们生成了大规模排列、经序列验证的文库，其中包含超过140,000个第二代短发夹RNA表达质粒，覆盖了人类和小鼠基因组中所有预测基因的很大一部分。这些文库可供科学界使用。
