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https://hdl.handle.net/1959.11/10128
Title: | Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of 'Ubiquitin-63E' in the cattle tick 'Rhipicephalus microplus' | Contributor(s): | Lew-Tabor, A E (author); Kurscheid, S (author); Barrero, R (author); Gondro, Cedric (author) ; Moolhuijzen, P M (author); Rodriguez Valle, M (author); Morgan, J A T (author); Covacin, C (author); Bellgard, M I (author) | Publication Date: | 2011 | DOI: | 10.1016/j.ijpara.2011.05.003 | Handle Link: | https://hdl.handle.net/1959.11/10128 | Abstract: | Knowledge of cattle tick ('Rhipicephalus (Boophilus) microplus'; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of ~50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the 'Drosophila Ubiquitin-63E' gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of 'R. microplus' ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick 'Ubiquitin-63E' 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 ('Ubiquitin-63E') were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp 'Ubiquitin-63E' dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting 'Ubiquitin-63E', demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (∼100-200 bp) and the usefulness of microarrays to study knockdown phenotypes. | Publication Type: | Journal Article | Source of Publication: | International Journal for Parasitology, 41(9), p. 1001-1014 | Publisher: | Elsevier Ltd | Place of Publication: | United Kingdom | ISSN: | 1879-0135 0020-7519 |
Fields of Research (FoR) 2008: | 070205 Animal Protection (Pests and Pathogens) | Socio-Economic Objective (SEO) 2008: | 830301 Beef Cattle | Peer Reviewed: | Yes | HERDC Category Description: | C1 Refereed Article in a Scholarly Journal |
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Appears in Collections: | Journal Article |
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