A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei
A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei
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DOI：
10.1016/j.molbiopara.2004.10.002
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发表时间：
2005-01-01
影响因子：
1.5
通讯作者：
Horn, D
中科院分区：
文献类型：
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作者：
Alibu, VP;Storm, L;Horn, D
在布鲁氏锥虫中产生条件突变最快的方法是使用RNA干扰。在带有LET操纵子的两个相反的T7启动子之间克隆靶序列的单个副本，所得到的质粒整合到同时表达LET阻遏子和T7 RNA聚合酶的细胞的基因组中。加入四环素后，由两个T7启动子合成双链RNA。不幸的是，对T7启动子活性的抑制有时可能不足以阻止有毒数量的双链RNA的表达。在这里，我们描述了T7聚合酶表达受四环素控制的细胞系，并证明了聚合酶表达的调节可以调节构成T7启动子的转录。此外，我们还描述了一种含有两个拷贝的m10Tet阻遏物的结构，以便于创建表达抑制物的锥体，以及一个RNA干扰载体，它允许对未修饰的PCR产物进行“TA”克隆和蓝/白选择。(C)2004爱思唯尔B.V.保留所有权利。
The most rapid method for the generation of conditional mutants in Trypanosoma brucei is the use of RNA interference. A single copy of the target sequence is cloned between two opposing T7 promoters bearing let operators, and the resulting plasmid is integrated into the genome of cells expressing both the let repressor and T7 RNA polymerase. Upon addition of tetracycline, double-stranded RNA is synthesised from the two T7 promoters. Unfortunately, repression of T7 promoter activity may sometimes be insufficient to prevent expression of toxic amounts of double-stranded RNA. We describe here cell lines in which the expression of T7 polymerase is under tetracycline control, and show that regulation of polymerase expression can modulate transcription from a constitutive T7 promoter. In addition we describe a construct containing two copies of the m 10 Tet repressor for easy creation of repressor-expressing trypanosomes, and an RNA interference vector which allows "TA" cloning of unmodified PCR products and blue/white selection. (C) 2004 Elsevier B.V. All rights reserved.