Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus

Lew-Tabor, A. E., Kurscheid, S., Barrero, R., Gondro, C., Moolhuijzen, P. M., Rodriguez Valle, M., Morgan, J. A. T., Covacin, C. and Bellgard, M. I. (2011) Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus. International Journal for Parasitology, 41 9: 1001-1014.

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Author Lew-Tabor, A. E.
Kurscheid, S.
Barrero, R.
Gondro, C.
Moolhuijzen, P. M.
Rodriguez Valle, M.
Morgan, J. A. T.
Covacin, C.
Bellgard, M. I.
Title Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus
Formatted title Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus
Journal name International Journal for Parasitology  (ERA 2012 Listed)    (ERA 2010 Rank A*)   Check publisher's open access policy
Publication date 2011-08
Sub-type Article
DOI 10.1016/j.ijpara.2011.05.003
Volume number 41
Issue number 9
ISSN 0020-7519; 1879-0135
Start page 1001
End page 1014
Total pages 14
Place of publication Oxford, United Kingdom
Publisher Elsevier
Collection year 2012
Language eng
Formatted 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.
Keyword RNA interference
Off-target effects
Ticks
Gene knockdown
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article
Collections: Official 2012 Collection
Queensland Alliance for Agriculture and Food Innovation
 
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