Inhibition of Trypanosoma brucei gene expression by RNA interference using an integratable vector with opposing T7 promoters
Inhibition of Trypanosoma brucei gene expression by RNA interference using an integratable vector with opposing T7 promoters
复制标题
利用带有对立 T7 启动子的可整合载体进行 RNA 干扰，抑制布氏锥虫基因的表达
DOI：
10.1074/jbc.m008405200
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发表时间：
2000-12-22
影响因子：
4.8
通讯作者：
Englund, PT
中科院分区：
文献类型：
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作者：
Wang, ZF;Morris, JC;Englund, PT
RNA干扰是抑制布氏锥虫基因表达的一种强有力的方法（Ngo, H., Tschudi, C,, Gull, K,, 和Ullu, E. (1998)《美国国家科学院院刊》95, 14687 - 14692）。在此我们描述了一种载体（pZJM），用于在稳定转化的细胞中体内四环素诱导合成双链RNA（dsRNA）。dsRNA由相对的T7启动子合成。我们用参与诸如动基体DNA复制、线粒体mRNA合成、糖基磷脂酰肌醇生物合成、糖质体生物发生以及多胺生物合成等过程的基因对该载体进行了测试。在大多数情况下，dsRNA的诱导导致相应mRNA特异性地大幅减少，并且在许多情况下出现生长抑制或细胞死亡。一种显著的表型是在干扰拓扑异构酶II的表达后动基体DNA丢失。通过这种方法分析的基因甚至不需要完全测序。事实上，我们测试的许多基因来自布氏锥虫基因组数据库中的部分序列，这些序列是通过与已知蛋白质的同源性鉴定出来的。从在数据库中鉴定一个基因序列到出现表型只需短短3周时间。
RNA interference is a powerful method for inhibition of gene expression in Trypanosoma brucei (Ngo, H., Tschudi, C,, Gull, K,, and Ullu, E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14687-14692), Here we describe a vector (pZJM) for in vivo tetracycline-inducible synthesis of double-stranded RNA (dsRNA) in stably transformed cells. The dsRNA is synthesized from opposing T7 promoters. We tested the vector with genes involved in processes such as kinetoplast DNA replication, mitochondrial mRNA synthesis, glycosyl phosphatidylinositol biosynthesis, glycosome biogenesis, and polyamine biosynthesis, In most cases the induction of dsRNA caused specific and dramatic loss of the appropriate mRNA and in many cases there was growth inhibition or cell death. One striking phenotype was the loss of kinetoplast DNA after interference with expression of a topoisomerase II. The gene being analyzed by this procedure need not even be fully sequenced. In fact, many of the genes we tested were derived from partial sequences in the T. brucei genome data base that were identified by homology with known proteins. It takes as little as 3 weeks from identification of a gene sequence in the data base to the appearance of a phenotype.