Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi

• Published in: Parasitology 2006-12, Vol.133 (6), p.777-783
• Main Authors: VISSER, A., GELDHOF, P., DE MAERE, V., KNOX, D. P., VERCRUYSSE, J., CLAEREBOUT, E.
• Format: Article
• Language: English
• Subjects: Animal populations; Animals; Biological and medical sciences; double-stranded RNA; Electroporation; Fundamental and applied biological sciences. Psychology; gastrointestinal nematodes; Gene expression; gene silencing; gene targeting; General aspects; General aspects and techniques. Study of several systematic groups. Models; Helminth Proteins - genetics; Helminth Proteins - metabolism; Immunization; Invertebrates; Larva - growth & development; Larvae; Life Cycle Stages; messenger RNA; Methods; nematode larvae; Ostertagia - genetics; Ostertagia - growth & development; Ostertagia - metabolism; Ostertagia ostertagi; Parasites; Protease inhibitors; Proteins; Research methodology; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA Interference; RNA polymerase; RNA, Helminth - genetics; RNA, Helminth - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; screening; Sensitivity and Specificity; soaking; Studies; Worms
• ISSN: 0031-1820; 1469-8161
• DOI: 10.1017/S0031182006001004

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, β-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.