Fast cloning inverted repeats for RNA interference

被引:20
作者
Bao, Sujin [1 ]
Cagan, Ross [1 ]
机构
[1] Washington Univ, Sch Med, Dept Mol Biol & Pharmacol, St Louis, MO 63110 USA
关键词
RNA interference; inverted repeat; cloning; Drosophila;
D O I
10.1261/rna.258406
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Double-stranded RNA ( dsRNA) can induce post-transcriptional gene silencing in a wide variety of organisms. Commonly, inverted repeats are used to produce dsRNA to silence genes of interest. However, cloning inverted repeats still remains a rate-limiting step for widely applying this technique. Here we describe a pGEM-T-based vector, pGEM-WIZ, designed to produce inverted repeats for any Drosophila gene. pGEM-WIZ has a high efficiency in assembling inverted repeats and the repeats in this vector are stable in regular Escherichia coli strains. Furthermore, we have developed a method for rapid selection of clones with an inverted repeat based on size and relative copy number of the vector with or without an insert. This method further eases the cloning process. The inverted repeat cassette assembled in pGEM-WIZ can be easily transferred to commonly available expression vectors suitable for stably expressing inverted repeats in vitro and in vivo.
引用
收藏
页码:2020 / 2024
页数:5
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