title: RNAi-mediated gene knockdown by microinjection in the model entomopathogenic nematode Heterorhabditis bacteriophora.
abstract: BACKGROUND
  Parasitic nematodes threaten the health of humans and livestock and cause a major financial and socioeconomic burden to modern society. Given the widespread distribution of diseases caused by parasitic nematodes there is an urgent need to develop tools that will elucidate the genetic complexity of host-parasite interactions. Heterorhabditis bacteriophora is a parasitic nematode that allows simultaneous monitoring of nematode infection processes and host immune function, and offers potential as a tractable model for parasitic nematode infections. However, molecular tools to investigate these processes are required prior to its widespread acceptance as a robust model organism. In this paper we describe microinjection in adult H. bacteriophora as a suitable means of dsRNA delivery to knockdown gene transcripts.
  
  
  METHODS
  RNA interference was used to knockdown four genes by injecting dsRNA directly into the gonad of adult hermaphrodite nematodes. RNAi phenotypes were scored in the F1 progeny on the fifth day post-injection, and knockdown of gene-specific transcripts was quantified with real-time quantitative RT-PCR (qRT-PCR).
  
  
  RESULTS
  RNAi injection in adult hermaphrodites significantly decreased the level of target transcripts to varying degrees when compared with controls. The genes targeted by RNAi via injection included cct-2, nol-5, dpy-7, and dpy-13. In each case, RNAi knockdown was confirmed phenotypically by examining the progeny of injected animals, and also confirmed at the transcriptional level by real-time qRT-PCR.
  
  
  CONCLUSIONS
  Here we describe for the first time the successful use of microinjection to knockdown gene transcripts in H. bacteriophora. This technique can be used widely to study the molecular basis of parasitism.
authors.affiliation: Department of Microbiology Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC, 20037, USA.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Ratnappan
authors.affiliation: Department of Microbiology Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC, 20037, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Vadnal
authors.affiliation: Department of Microbiology Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC, 20037, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Keaney
authors.affiliation: Department of Biological Sciences, George Washington University, Science and Engineering Hall, suite 6000, 800 22nd Street NW, Washington, DC, 20052, USA.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Eleftherianos
authors.affiliation: Department of Biological Sciences, George Washington University, Science and Engineering Hall, suite 6000, 800 22nd Street NW, Washington, DC, 20052, USA.|Institute for Neuroscience, George Washington University, 636 Ross Hall, 2300 I Street NW, Washington, DC, 20052, USA.
authors.forename: Damien
authors.initials: D
authors.lastname: O'Halloran
authors.affiliation: Department of Microbiology Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC, 20037, USA. jhawdon@gwu.edu.
authors.forename: John M
authors.initials: JM
authors.lastname: Hawdon
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques / Q000379:methods*; D008845:Microinjections / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D017172:Rhabditoidea / Q000235:genetics*
keywords: Gene knockdown; Heterorhabditis bacteriophora; Microinjection; RNAi; qRT-PCR