tailieunhanh - Báo cáo khoa học: Design of expression vectors for RNA interference based on miRNAs and RNA splicing

RNA interference (RNAi) mediates sequence-specific post-transcriptional gene silencing in many eukaryotes and is used for reverse genetic studies and therapeutics. RNAi is triggered by double-stranded small interfering RNAs (siRNAs), which can be processed from small hairpin RNAs gener-ated from an expression vector. | ỊFEBS Journal Design of expression vectors for RNA interference based on miRNAs and RNA splicing Guangwei Du Joshua Yonekubo Yue Zeng Mary Osisami and Michael A. Frohman Department of Pharmacology and the Center for DevelopmentalGenetics Stony Brook University NY USA Keywords intron miRNA RNA interference RNA splicing small-hairpin RNA Correspondence G. Du Department of Pharmacology and the Center for DevelopmentalGenetics Stony Brook University Stony Brook NY 11794-5140 USA Fax 1 631 632 1692 Tel 1 631 632 1477 E-mail guangwei@ Received 13 July 2006 revised 9 September 2006 accepted 11 October 2006 doi RNA interference RNAi mediates sequence-specific post-transcriptional gene silencing in many eukaryotes and is used for reverse genetic studies and therapeutics. RNAi is triggered by double-stranded small interfering RNAs siRNAs which can be processed from small hairpin RNAs generated from an expression vector. In some recently described vectors the siRNAs are expressed from synthetic stem-loop precursors of microRNAs miRNAs driven by polymerase II promoters. We have designed new RNAi vectors designated pSM155 and pSM30 that take into consideration miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. Like the original miRNA vectors we show that the pSM155 and pSM30 constructs efficiently down-regulate expression of firefly luciferase and an endogenous gene phospholipase D2. Moreover the expression of a coexpressed fluorescent marker is substantially improved by this new design. Another improvement of these new vectors is incorporation of two inverted Bsm BI sites placed internal to the arms of the new miRNA-based vectors so oligos used for cloning are shorter and the cost is reduced. These RNAi vectors thus provide new tools for gene suppression. Target genes can be silenced by transfection of chemically or enzymatically synthesized small .