Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica.
ABSTRACT Experimental procedures using the RNA interference (RNAi) approach have recently emerged as a powerful tool for gene silencing in eukaryotic microbes for which gene replacement techniques have not yet been developed. Our group has recently explored RNAi to knock down gene-specific expression in the protozoan parasite Entamoeba histolytica, through delivery of small interfering RNA (siRNA) oligonucleotides by the soaking approach. Standardized conditions for the soaking of E. histolytica trophozoites with siRNAs result in highly specific and significant silencing of parasite cognate genes. Real-time PCR analysis indicates that a 16-hour treatment with siRNAs usually results in half-extinction of target messenger RNA. Furthermore, Western blot analysis of trophozoite crude extracts with the use of specific antibodies shows a similar reduction of cognate protein levels after siRNA treatment.
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ABSTRACT: Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.Journal of Visualized Experiments 01/2010; DOI:10.3791/2410
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ABSTRACT: In many eukaryotes, the introduction of double-stranded RNA (dsRNA) into cells triggers the degradation of mRNAs through a post-transcriptional gene-silencing mechanism called RNA interference or RNAi. In the present study, we found that endogenous long-dsRNA was substantially more effective at producing interference than endogenous, or exogenous, short-dsRNA expression in Giardia lamblia . The effects of this interference were not evident in the highly expressed protein tubulin or the stage-specific cyst wall protein 2. However, long-dsRNA caused potent and specific interference in the medium subunits of adaptins, the RNA-dependent RNA polymerase, and the exogenous green fluorescence protein. Our results suggest that the ability of dsRNA antisense to inhibit the expression of these specific types of proteins is indicative of a gene-specific mechanism.Journal of Parasitology 08/2010; 96(4):815-9. DOI:10.1645/GE-2406.1 · 1.26 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs regulating gene expression in eukaryotes at the post-transcriptional level. The complex life cycles of parasites may require the ability to respond to environmental and developmental signals through miRNA-mediated gene expression. Over the past 17 years, thousands of miRNAs have been identified in the nematode Caenorhabditis elegans and other parasites. Here, we review the current status and potential functions of miRNAs in protozoan, helminths, and arthropods, and propose some perspectives for future studies.Parasitology Research 08/2010; 107(3):501-7. DOI:10.1007/s00436-010-1927-6 · 2.33 Impact Factor