title: Direct experimental manipulation of intestinal cells in Ascaris suum, with minor influences on the global transcriptome.
abstract: Ascaris suum provides a powerful model for studying parasitic nematodes, including individual tissues such as the intestine, an established target for anthelmintic treatments. Here, we add a valuable experimental component to our existing functional, proteomic, transcriptomic and phylogenomic studies of the Ascaris suum intestine, by developing a method to manipulate intestinal cell functions via direct delivery of experimental treatments (in this case, double-stranded (ds)RNA) to the apical intestinal membrane. We developed an intestinal perfusion method for direct, controlled delivery of dsRNA/heterogeneous small interfering (hsi) RNA into the intestinal lumen for experimentation. RNA-Seq (22 samples) was used to assess influences of the method on global intestinal gene expression. Successful mRNA-specific knockdown in intestinal cells of adult A. suum was accomplished with this new experimental method. Global transcriptional profiling confirmed that targeted transcripts were knocked down more significantly than any others, with only 12 (0.07% of all genes) or 238 (1.3%) off-target gene transcripts consistently differentially regulated by dsRNA treatment or the perfusion experimental design, respectively (after 24h). The system supports controlled, effective delivery of treatments (dsRNA/hsiRNA) to the apical intestinal membrane with relatively minor off-target effects, and builds on our experimental model to dissect A. suum intestinal cell functions with broad relevance to parasitic nematodes.
authors.affiliation: The McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Rosa
authors.affiliation: The McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Samantha N
authors.initials: SN
authors.lastname: McNulty
authors.affiliation: The McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA; Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Genetics, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Makedonka
authors.initials: M
authors.lastname: Mitreva
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA. Electronic address: djasmer@vetmed.wsu.edu.
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Jasmer
mesh_terms: D000818:Animals; D001196:Ascariasis / Q000469:parasitology; D017165:Ascaris suum / Q000166:cytology / Q000235:genetics*; D001483:Base Sequence; D005260:Female; D055785:Gene Knockdown Techniques; D049751:Genome, Helminth; D007422:Intestines / Q000187:drug effects / Q000502:physiology; D008297:Male; D010802:Phylogeny; D012330:RNA, Double-Stranded / Q000008:administration & dosage* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013552:Swine; D059467:Transcriptome
chemical_list: D012330:RNA, Double-Stranded; D012333:RNA, Messenger
keywords: A. suum; Intestine; Methodology; Nematode; RNAi; dsRNA