Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells
Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells
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DOI：
10.1038/nmeth1005-779
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发表时间：
2005-10-01
期刊：
影响因子：
48
通讯作者：
Buchholz, F
中科院分区：
文献类型：
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作者：
Kittler, R;Heninger, AK;Buchholz, F
RNA干扰（RNAi）已经彻底改变了许多模式生物的功能基因组研究。特别是，在哺乳动物细胞中引入短双链rna （dsRNAs）作为RNAi的介质，将这些实验系统中的功能丧失研究推向了一个新的层面(1)。目前，哺乳动物细胞中最常用的两种RNAi触发器是化学或体外合成的短干扰rna (sirna)(2)和体内表达的短发夹rna (shRNAs)(3)。我们和其他人开创了一种替代技术，这种技术比siRNA和shRNA方法更具成本效益，也更省力。我们的方法是利用重组大肠杆菌RNase III或Dicer酶切Long dsRNAs产生sirna（4-8）。由此产生的核糖核酸内酶制备的sirna （esiRNAs）已被证明是哺乳动物细胞中RNAi的高效和特异性介质（9-11）。在这里，我们描述了一种强大而简单的esiRNA生产方案（图1）：通过PCR标记T7启动子序列的cDNA片段在体外转录以产生dsRNA。通过有限的消化，长dsRNA被转化为小于30个碱基对（bp）的sirna，并在单柱中进行自旋纯化。由于其简单，快速和成本效益，该协议可以很容易地扩展到（亚）基因组筛选文库的生成。
RNA interference (RNAi) has revolutionized functional genomic Studies in many model organisms. In particular, the introduction of short double-stranded RNAs (dsRNAs) as mediators of RNAi in mammalian cells has moved Loss-of-function studies in these experimental systems to a new plane(1). Now the two most commonly used RNAi triggers in mammalian cells are chemically or in vitro synthesized short interfering RNAs (siRNAs)(2) and in vivo expressed short hairpin RNAs (shRNAs)(3). We, and others, have pioneered an alternative technology, which is considerably more cost-efficient and less Laborious than siRNA and shRNA methodologies. Our method is based on the generation of siRNAs by digestion of Long dsRNAs with recombinant Escherichia coli RNase III or Dicer(4-8). The resulting endoribonuclease-prepared siRNAs (esiRNAs) have proven to be efficient and specific mediators of RNAi in mammalian cells(9-11). Here we describe a robust and simple protocol for the production of esiRNA (Fig. 1): a cDNA fragment tagged with T7 promoter sequence by PCR is transcribed in vitro to produce dsRNA. By limited digestion, the tong dsRNA is then converted to siRNAs of < 30 base pairs (bp) and spin-purified in a single column. Because of its ease, speed and cost-efficiency, this protocol can be easily scaled for the generation of libraries for (sub)genomic screens.