Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli.
Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli.
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DOI：
10.1093/nar/gkp498
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发表时间：
2009-08
影响因子：
14.9
通讯作者：
Tamura T
中科院分区：
文献类型：
--
作者：
Nakashima N;Tamura T
在这项研究中，我们描述了一种利用反义RNA同时对大肠杆菌中多达四个基因进行条件性基因沉默的方法。我们使用带有成对末端的反义RNA，它携带侧翼反向重复序列来创建成对的双链RNA末端；这些RNA已被证明具有很高的沉默效率。为了表达反义RNA，我们构建了四个携带不同但相容的复制起点的IPTG诱导载体。当用这些载体表达LacZ反义RNA时，所有载体都成功地沉默了LacZ的表达，但反义RNA的表达水平和沉默效果因载体的不同而不同。所有载体都是可共转化的，针对LacZ、ackA、PTA和PepN的反义RNA共表达，证实了所有目的基因的沉默。此外，当反义RNA针对参与DNA复制或DNA错配修复的突变基因MutS、muD(Dna Q)和NDK时，自发突变频率增加了2000倍以上。由此产生的突变株可用于随机诱变质粒。该方法为研究多个基因之间的功能关系或改变生物技术和工业应用的细胞表型提供了一个强大的工具。
In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been proven to have high silencing efficacy. To express antisense RNAs, we constructed four IPTG-inducible vectors carrying different but compatible replication origins. When the lacZ antisense RNA was expressed using these vectors, lacZ expression was successfully silenced by all the vectors, but the expression level of the antisense RNA and silencing efficacy differed depending on the used vectors. All the vectors were co-transformable; the antisense RNAs against lacZ, ackA, pta and pepN were co-expressed, and silencing of all the target genes was confirmed. Furthermore, when antisense RNAs were targeted to the mutator genes mutS, mutD (dnaQ) and ndk, which are involved in DNA replication or DNA mismatch repair, spontaneous mutation frequencies increased over 2000-fold. The resulting mutator strain is useful for random mutagenesis of plasmids. The method provides a robust tool for investigating functional relationships between multiple genes or altering cell phenotypes for biotechnological and industrial applications.
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影响因子：
3.6
作者：
BLOMFIELD, IC;VAUGHN, V;EISENSTEIN, BI
通讯作者：
EISENSTEIN, BI
影响因子：
14.9
作者：
Blokpoel MC;Murphy HN;O'Toole R;Wiles S;Runn ES;Stewart GR;Young DB;Robertson BD
通讯作者：
Robertson BD
影响因子：
14.9
作者：
Calmann MA;Evans JE;Marinus MG
通讯作者：
Marinus MG
影响因子：
16
作者：
Bouvier, Marie;Sharma, Cynthia M.;Vogel, Joerg
通讯作者：
Vogel, Joerg
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL