title: RNA interference in Fasciola hepatica newly excysted juveniles: long dsRNA induces more persistent silencing than siRNA.
abstract: In trematodes RNA interference is the current tool of choice for functional analysis of genes since classical reverse genetic approaches remain unavailable. Whereas this approach has been optimized in schistosomes, few reports are available for other trematodes, likely reflecting the difficulties in the establishment of the technology. Here we standardized conditions for RNAi in the liver fluke Fasciola hepatica, the causative agent of fasciolosis, one of the most problematic infections affecting livestock worldwide. Targeting a single copy gene, encoding leucine aminopeptidase (LAP) as a model, we refined delivery conditions which identified electro-soaking, i.e. electroporation and subsequent incubation as efficient for introduction of small RNAs into the fluke. Knock down of LAP was achieved with as little as 2.5 μg/ml dsRNA concentrations, which may reduce or obviate off-target effects. However, at these concentrations, tracking incorporation by fluorescent labeling was difficult. While both long dsRNA and short interfering RNA (siRNA) are equally effective at inducing a short-term knock down, dsRNA induced persistent silencing up to 21 days after treatment, suggesting that mechanisms of amplification of the interfering signal can be present in this pathogen. Persistent silencing of the invasive stage for up to 3 weeks (close to what it takes for the fluke to reach the liver) opens the possibility of using RNAi for the validation of putative therapeutic targets.
authors.affiliation: Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), Gral. Flores 2125, CP 11800 Montevideo, Uruguay. Electronic address: ndelloca@fmed.edu.uy.
authors.forename: Nicolás
authors.initials: N
authors.lastname: Dell'Oca
authors.affiliation: Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), Gral. Flores 2125, CP 11800 Montevideo, Uruguay. Electronic address: tbasika@gmail.com.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Basika
authors.affiliation: Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), Gral. Flores 2125, CP 11800 Montevideo, Uruguay. Electronic address: icorvo@fmed.edu.uy.
authors.forename: Ileana
authors.initials: I
authors.lastname: Corvo
authors.affiliation: Sección Bioquímica, Facultad de Ciencias, Universidad de la Republica (UDELAR), Iguá 4225, CP 11400 Montevideo, Uruguay. Electronic address: castillo@fcien.edu.uy.
authors.forename: Estela
authors.initials: E
authors.lastname: Castillo
authors.affiliation: Department of Microbiology, Immunology and Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, The George Washington University, 2300 I Street NW, Washington, DC 20037, USA. Electronic address: pbrindley@gwu.edu.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brindley
authors.affiliation: Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), Gral. Flores 2125, CP 11800 Montevideo, Uruguay; Department of Microbiology, Immunology and Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, The George Washington University, 2300 I Street NW, Washington, DC 20037, USA. Electronic address: grinaldi@gwu.edu.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Rinaldi
authors.affiliation: Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), Gral. Flores 2125, CP 11800 Montevideo, Uruguay. Electronic address: jtort@fmed.edu.uy.
authors.forename: Jose F
authors.initials: JF
authors.lastname: Tort
mesh_terms: D000818:Animals; D005210:Fasciola hepatica / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering
keywords: Fasciola; RNAi delivery methods; RNAi silencing; Trematodes; siRNA