Fast cloning inverted repeats for RNA interference
Fast cloning inverted repeats for RNA interference
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DOI：
10.1261/rna.258406
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发表时间：
2006-11-01
期刊：
影响因子：
4.5
通讯作者：
Cagan, Ross
中科院分区：
文献类型：
--
作者：
Bao, Sujin;Cagan, Ross
双链RNA(Double-stranded RNA，dsRNA)可诱导多种生物体内转录后基因沉默。通常，反向重复序列被用来产生dsRNA来沉默感兴趣的基因。然而，克隆反向重复序列仍然是广泛应用这项技术的一个速率限制步骤。在这里，我们描述了一种基于pGEM-T的载体pGEM-WIZ，旨在为任何果蝇基因产生反向重复序列。PGEM-WIZ在组装反向重复序列方面效率很高，并且该载体中的重复序列在正常的大肠杆菌菌株中是稳定的。此外，我们还开发了一种基于有或没有插入的载体的大小和相对拷贝数来快速选择具有反向重复的克隆的方法。这种方法进一步简化了克隆过程。在pGEM-WIZ中组装的反向重复序列盒可以很容易地转移到适合在体内外稳定表达反向重复序列的常用表达载体上。
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.