A miR-21 hairpin structure-based gene knockdown vector.
A miR-21 hairpin structure-based gene knockdown vector.
复制标题
miR-21发夹结构基因敲低载体。
DOI：
10.1016/j.bbrc.2010.03.047
复制
发表时间：
2010-04-09
影响因子：
3.1
通讯作者：
Penmatsa, Sravya
中科院分区：
文献类型：
--
作者：
Yue, Junming;Sheng, Yi;Ren, Aixia;Penmatsa, Sravya
RNA干扰（RNA interference，RNAi）作为从第一代siRNA到第二代短发夹RNA（short hairpin RNA，shRNA）或新开发的microRNA（shRNA-miR）的反向遗传学手段，被广泛用于基因功能研究。本文报道了一种基于小鼠miR-21发夹结构的基因敲除载体系统。在该系统中，miR-21基因的pre-miRNA发夹通过用靶向感兴趣基因的shRNA序列替换22个核苷酸的成熟序列进行修饰，所述shRNA序列侧接小鼠pre-miR-21的160-bp上游和65-bp下游序列。我们通过使用不同的载体敲除增强型绿色荧光蛋白（EGFP）报告基因来测试该系统，其中shRNA-miR由聚合酶II（pol II）启动子驱动。我们发现，基于miR-21发夹的shRNA-miR可以直接置于pol II启动子下，如UbC或CMV启动子，以敲低目的基因。为了促进基于miR-21发夹的基因敲除系统的广泛应用，我们进一步敲除内源性核纤层蛋白（A/C），这表明使用基于miR-21发夹的慢病毒载体可以有效地沉默内源性核纤层蛋白A/C表达。基于miR-21发夹结构的基因敲减载体将为体内外基因功能研究提供新的遗传工具。
RNA interference (RNAi) is widely used to study gene functions as a reverse genetic means from first-generation siRNA to second-generation short hairpin RNA (shRNA) or the newly developed microRNA (shRNA-miR). Here we report a gene knockdown vector system based on the mouse miR-21 hairpin structure. In this system, the pre-miRNA hairpin of the miR-21 gene was modified by replacing the 22-nucleotide mature sequence with shRNA sequences that target genes of interest, flanked by 160-bp upstream and 65-bp downstream sequences of the mouse pre-miR-21. We tested this system by knocking down the enhanced green fluorescence protein (EGFP) reporter gene using different vectors, in which shRNA-miR was driven by the polymerase II (pol II) promoter. We found that miR-21 hairpin-based the shRNA-miR can be directly placed under pol II promoter, like UbC or CMV promoter to knockdown the gene of interest. To facilitate the wide application of the miR-21hairpin-based gene knockdown system, we further knocked down the endogenous gene lamin (A/C), which showed that endogenous lamin A/C expression can be efficiently silenced using the miR-21 hairpin -based lentiviral vector. The miR-21hairpin-based gene knockdown vector will provide a new genetic tool for gene functional studies in vitro and in vivo.
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影响因子：
4.5
作者：
Xia XG;Zhou H;Samper E;Melov S;Xu Z
通讯作者：
Xu Z
影响因子：
5.1
作者：
Bauer, M.;Kinkl, N.;Ueffing, M.
通讯作者：
Ueffing, M.
DOI：
10.1073/pnas.0604657103
发表时间：
2006-07-25
影响因子：
11.1
作者：
Dann, Christina Tenenhaus;Alvarado, Alma L.;Garbers, David L.
通讯作者：
Garbers, David L.
影响因子：
30.8
作者：
Silva, JM;Li, MZ;Hannon, GJ
通讯作者：
Hannon, GJ
影响因子：
12.4
作者：
Boudreau, Ryan L.;Martins, Ines;Davidson, Beverly L.
通讯作者：
Davidson, Beverly L.