Second-generation shRNA libraries covering the mouse and human genomes
Second-generation shRNA libraries covering the mouse and human genomes
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DOI：
10.1038/ng1650
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发表时间：
2005-11-01
期刊：
影响因子：
30.8
通讯作者：
Hannon, GJ
中科院分区：
文献类型：
--
作者：
Silva, JM;Li, MZ;Hannon, GJ
功能丧失表型通常是理解生物化学途径的连接和生物学功能的关键。我们和其他人先前构建了短发夹RNA文库，可以系统分析哺乳动物细胞中RNA干扰诱导的表型。在这里，我们报告的第二代短发夹RNA表达文库的构建和验证设计使用RNA干扰生物化学的知识增加。这些构建体包括设计用于模拟天然微小RNA初级转录物的沉默触发物，并且基于最佳小RNA性能的热力学标准选择每个靶序列。生物化学和表型分析表明，新的图书馆大大改善了第一代试剂。我们生成了大规模阵列的序列验证文库，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。这些图书馆可供科学界使用。
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.