title: Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi.
abstract: BACKGROUND
  RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans.
  
  
  RESULTS
  We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week.
  
  
  CONCLUSIONS
  We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95604, USA. landmann@biology.ucsc.edu
authors.forename: Frédéric
authors.initials: F
authors.lastname: Landmann
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Foster
authors.forename: Barton E
authors.initials: BE
authors.lastname: Slatko
authors.forename: William
authors.initials: W
authors.lastname: Sullivan
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000196:embryology / Q000235:genetics* / Q000648:ultrastructure; D017173:Caenorhabditis elegans / Q000235:genetics; D005260:Female; D005368:Filariasis / Q000469:parasitology*; D005455:Fluorescent Antibody Technique; D017238:Genes, Helminth / Q000235:genetics; D006801:Humans; D010641:Phenotype; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D018510:RNA, Helminth / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D012330:RNA, Double-Stranded; D018510:RNA, Helminth