Enzymatic production of RNAi libraries from cDNAs
Enzymatic production of RNAi libraries from cDNAs
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DOI：
10.1038/ng1290
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发表时间：
2004-02-01
期刊：
影响因子：
30.8
通讯作者：
Hirose, K
中科院分区：
文献类型：
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作者：
Shirane, D;Sugao, K;Hirose, K
由小干扰(SiRNA)或短发夹状RNA(ShRNA)诱导的RNA干扰(RNAi)是哺乳动物遗传学的一种重要研究方法。在这里，我们描述了一种称为RNAi文库的酶生产(EPRIL)技术，通过该技术，一系列酶处理将cDNA转化为由大量不同shRNA表达结构组成的RNAi文库。我们将EPRIL应用于单一的cDNA源，并制备了由具有不同RNAi效率的shRNA构建的RNAi文库。高通量筛选使我们能够快速从文库中鉴定出最好的shRNA构建体。我们还描述了一种使用胸苷激酶基因来获得有效shRNA构建体的新的选择方案。此外，我们还证明了EPRIL可以应用于从cDNA文库构建RNAi文库，为未来全基因组的基因表型筛选提供了基础。
RNA interference (RNAi) induced by small interfering (siRNA) or short hairpin RNA (shRNA) is an important research approach in mammalian genetics. Here we describe a technology called enzymatic production of RNAi library (EPRIL) by which cDNAs are converted by a sequence of enzymatic treatments into an RNAi library consisting of a vast array of different shRNA expression constructs. We applied EPRIL to a single cDNA source and prepared an RNAi library consisting of shRNA constructs with various RNAi efficiencies. High-throughput screening allowed us to rapidly identify the best shRNA constructs from the library. We also describe a new selection scheme using the thymidine kinase gene for obtaining efficient shRNA constructs. Furthermore, we show that EPRIL can be applied to constructing an RNAi library from a cDNA library, providing a basis for future whole-genome phenotypic screening of genes.