RNA interference in Haemonchus contortus: suppression of beta-tubulin gene expression in L3, L4 and adult worms in vitro.
We have used RNAi to target two beta-tubulin genes in the parasitic stages of Haemonchus contortus in vitro. Soaking exsheathed-L3, L4 and adult worms in medium containing dsRNA resulted in a significant decrease (greater than 1000-fold in some cases) in the expression of the specific beta-tubulin transcript, as measured by quantitative PCR. During the initial 24h exposure to the dsRNA, the gene suppression effect was quite specific to the targeted gene. Six days after initial exposure to dsRNA, treated L3 worms were less able to migrate through a filter mesh, indicating decreased motility, and showed less development to the L4 stage than control larvae. The gene suppression effect occurs in exsheathed L3 larvae despite the fact that this life stage does not have functioning mouthparts, indicating that uptake of dsRNA does not depend on its ingestion. Suppression occurred with dsRNA presented in 'naked' or liposome-encapsulated forms, indicating that a liposome formulation was not necessary for uptake to occur. Adult worms also showed significant gene suppression, however, they did not show any reduced motility compared to controls over a 3-day period. Adult worms treated with ivermectin to paralyse their pharynx, still showed significant gene suppression, again suggesting that uptake of dsRNA does not require ingestion. We have shown that soaking in dsRNA is an effective method for RNAi with the parasitic stages of H. contortus, and, hence, may offer significant potential as a tool for studying gene function in this parasite species.
Kotze
Andrew C
AC
CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia, Qld 4067, Australia. andrew.kotze@csiro.au
Bagnall
Neil H
NH
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry; D006189:Haemonchus / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism*; D018510:RNA, Helminth / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D014404:Tubulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D012330:RNA, Double-Stranded; D018510:RNA, Helminth; D012333:RNA, Messenger; D014404:Tubulin