Silencing Genes by RNA Interference in the Protozoan Parasite Entamoeba histolytica

Unité de Biologie Cellulaire du Parasitisme, Institut Pasteur, Paris, France.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2008; 442:113-28. DOI: 10.1007/978-1-59745-191-8_9
Source: PubMed


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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    • "Key to the utilisation of gene-silencing methods is the presence of complete RNA-interference (RNAi) machinery in the parasite. Essential components of this pathway (e.g., DICER) have been well explored in Giardia (Macrae et al., 2006; Prucca et al., 2008), and RNAi has been used for gene perturbation for G. intestinalis (see Gargantini et al., 2011) and E. histolytica (see Solis and Guillen, 2008), suggesting that RNAi pathways are sufficiently complete in both of these organisms. Sequencing of the C. parvum and C. hominis genomes has revealed a lack of identifiable homologues to DICER and Argonaute, suggesting an altered and/or absent role for small interferring RNAs in gene regulation in these organisms (Abrahamsen et al., 2004; Xu et al., 2004), indicating against the use of RNAi technologies for functional exploration of Cryptosporidium . "
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    • "The siRNA-A with the scrambled sequence was used at 40 μg per culture following the manufacturer's instructions. Transfection of the trophozoites was performed by soaking as previously reported [17]. In brief, 1 × 105 trophozoites were grown in 6 mL TYI-S33 media in culture flasks. "
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