Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei

被引:49
作者
Durand-Dubief, M
Kohl, L
Bastin, P
机构
[1] Museum Matl Hist Nat, CNRS, UMR 8646, Lab Biophys, F-75231 Paris 05, France
[2] Unite INSERM U565, F-75231 Paris 05, France
关键词
Trypanosoma; RNA interference; flagellum; phenotype; FUNCTIONAL GENOMIC ANALYSIS; GERM-LINE DEVELOPMENT; PARAFLAGELLAR ROD; C-ELEGANS; GENETIC INTERFERENCE; MAMMALIAN-CELLS; MESSENGER-RNA; VIRUS-RESISTANCE; ANTISENSE RNA; EXPRESSION;
D O I
10.1016/S0166-6851(03)00071-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
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页码:11 / 21
页数:11
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