Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei
Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei
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DOI：
10.1016/s0166-6851(03)00071-9
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发表时间：
2003-06-01
影响因子：
1.5
通讯作者：
Bastin, P
中科院分区：
文献类型：
--
作者：
Durand-Dubief, M;Kohl, L;Bastin, P
在许多真核生物中。双链（ds）RNA导致同源序列的RNA的特异性降解，该过程称为RNA干扰（RNAi）。在这里，我们使用原生动物布氏锥虫作为模型，以调查的效率和特异性的RNAi产生的长dsRNA的PFRA和PFRC基因，编码鞭毛蛋白所需的细胞运动的表达。使用群体或个体细胞分析，在所有三个水平监测RNAi的后果：靶RNA表达、蛋白质表达和表型观察。来自反向重复序列的PFRA双链RNA的表达非常有效，敲除PFRA RNA和PFRA蛋白，并产生严重的瘫痪表型。通过使用双向启动子系统表达PFRA dsRNA进行沉默也是非常有效的，产生了明确的表型，尽管检测到少量的PFRA RNA和PFRA蛋白。通过PAR 2 dsRNA（与PFRA的83%总体同一性，包括9个>20 nt总体同一性的嵌段）的双面向启动子的表达不产生PFRA RNA或PFRA蛋白总量的显著减少。然而，通过免疫荧光的单个细胞分析显示，10-60%的细胞（取决于亚克隆）在其鞭毛中表现出较低的PFRA量，产生运动性降低的表型。(C)2003 Elsevier Science B. V.保留所有权利。
In many eukaryotes. double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10-60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype. (C) 2003 Elsevier Science B.V. All rights reserved.