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KMID : 0381120130350030395
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Genes and Genomics
2013 Volume.35 No. 3 p.395 ~ p.403
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Enzymatic production and expression of shRNAmir30 from cDNAs
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Kim Min-Jung
Park Seong-Kyun
Choi Vit-Na
Seo Dong-Wan
Lee Seung-Ho
Kee Yun
Hwang Byung-Joon
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Abstract
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RNA interference (RNAi) is an important tool for studying gene function and genetic networks. Double-stranded RNA (dsRNA) triggers RNAi that selectively silences gene expression mainly by degrading target mRNA sequences. Short interfering RNA, short hairpin RNA (shRNA), long dsRNA, and microRNA-based shRNA (shRNAmir) are four different types of dsRNA that have been widely used to silence gene expression in cultured cells, tissues, organs, and organisms. Long dsRNAs are usually 200?500 nucleotides in length and can selectively suppress expression of target genes in Caenorhabditis elegans and Drosophila but not in mammals due to unwanted non-specific knockdown. Thus, multiple attempts have been made to synthesize, express, and deliver short dsRNAs that specifically silence target genes in mammals. We describe a method for constructing an RNAi library by converting cDNAs into shRNAmir30 sequences by sequential treatment with different enzymes and affinity purification of biotin- or digoxygenin-labeled DNA fragments. We also developed a system to generate stable cell lines that uniformly express shRNAmir30s and fluorescence reporters by Cre recombinase-dependent site-specific recombination. Thus, combined with the RNAi library, this system facilitates screening for potent RNAi sequences that strongly suppress expression of target genes.
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KEYWORD
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RNAi, shRNA, Cre recombinase
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