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BMC Microbiology 2009
Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolyticaAbstract: An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%.Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.The human parasite Entamoeba histolytica (E. histolytica) is a unicellular protozoal parasite that phylogenetically is placed on one of the lowermost branches of the eukaryotic tree, closest to Dictyostelium discoideum [1]. It is an unusual organism, having 9,938 predicted genes, with slightly less than one third (31.8%) of its predicted proteins having no homologues in GenBank [2]. Humans are its only natural hosts, and E. histolytica is spread by ingestion of contaminated food or water via the fecal-oral route and thus tends to endemically infect people under circumstances where hygiene is poor [3]. It has a simple life cycle, alternating between infective quadrinucleate cysts and invasive motile trophozoites [3]. 80% of people infected with E. histolytica are colonized asymptomatically; in the remaining 20%, trophozoites invade into the intestinal epithelium, resulting in clinical disease [3]. It is estimated that there are 50 million symptomatic cases of amebic colitis and 100,000 deaths per year worldwide due to E. histolytica [4].The discovery that double-stranded RNA (dsRNA) can initiate post-transcriptional sequence-specific gene silencing of cellular genes [5] via translational repression or degradat
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