Testing the efficacy of RNA interference in Haemonchus contortus.
RNA interference (RNAi) is widely used in Caenorhabditis elegans to identify gene function and has been adapted as a high throughput screening method to identify genes involved in essential processes. We have been examining whether RNAi could also be used on the strongylid parasitic nematode Haemonchus contortus to study gene function. Eleven genes were targeted in L1 and exsheathed L3 H. contortus larvae with RNAi methodologies which have been shown to be effective in C. elegans and parasitic nematodes-feeding, soaking and electroporation. Reverse transcriptase-PCR and, where possible, protein assays were carried out to examine decreases in mRNA and protein levels. RNAi soaking in dsRNA to beta-tubulin and sec-23, a gene involved in vesicle transport, resulted in specific decreases in mRNA levels in exsheathed L3 larvae. No signs of specific decreases in expression levels were observed for the other nine genes tested. Following electroporation of dsRNA in L1 stage larvae, significant decreases were observed for two out of four genes tested. These findings suggest that the RNAi pathway is functional in H. contortus and that, under certain conditions, it is possible to suppress gene expression by RNAi. However, it only works on a limited number of genes and in some cases the effect is small and difficult to reproduce. This indicates that the RNAi approaches established for C. elegans and other nematodes have limited efficacy in H. contortus. This may reflect differences between nematode species in dsRNA uptake and transport into cells and between cells.
Moredun Research Institute, Pentlands Science Park, Penicuik, Midlothian EH26 0PZ, UK.
Peter
P
Geldhof
Linda
L
Murray
Annabelle
A
Couthier
John S
JS
Gilleard
Gerard
G
McLauchlan
David P
DP
Knox
Collette
C
Britton
D000818:Animals; D001483:Base Sequence; D004435:Eating / Q000235:genetics; D018274:Electroporation; D005786:Gene Expression Regulation / Q000235:genetics; D020868:Gene Silencing / Q000502:physiology; D017238:Genes, Helminth; D006189:Haemonchus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D018510:RNA, Helminth / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D015801:Helminth Proteins; D012330:RNA, Double-Stranded; D018510:RNA, Helminth; D012333:RNA, Messenger