Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors
Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors
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DOI：
10.1111/lam.12066
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发表时间：
2013-06-01
影响因子：
2.4
通讯作者：
Tamura, T.
中科院分区：
文献类型：
--
作者：
Nakashima, N.;Tamura, T.
在这里，我们报告了在大肠杆菌中表达反义 RNA 的多西环素（四环素类似物）诱导型载体的构建。使用这些载体，可以有条件地沉默感兴趣基因的表达。与之前构建的异丙基-D-吡喃半乳糖苷诱导型载体相比，该载体的反义RNA表达受到更严格的调控。此外，通过将多西环素诱导型启动子与T7启动子-T7 RNA聚合酶系统相结合，反义RNA的表达水平得到增强。 T7 RNA 聚合酶基因在多西环素诱导型启动子的控制下，被整合到基因组的 lacZ 基因座中，而没有留下任何抗生素标记。这些载体可用于研究基因功能或改变生物技术和工业应用的细胞表型。
Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl--D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications.