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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. and Kurscheid, S. and Barrero, R. and Gondro, C. and Moolhuijzen, P.M. and Valle, M.R. and Morgan, J.A.T. and 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). pp. 1001-1014.

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Article Link(s): http://dx.doi.org/10.1016/j.ijpara.2011.05.003

Publisher URL: http://www.elsevier.com

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 similar to 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 (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Item Type:Article
Corporate Creators:Department of Employment, Economic Development & Innovations (DEEDI), Queensland Alliance for Agriculture, Food and Innovation (QAFFI), Agri-Science, Animal Science
Business groups:Agri-Science, Animal Science, QAFFI
Additional Information:© Australian Society for Parasitology Inc.
Keywords:RNA interference; off-target effects; ticks; gene knockdown; boophilus-microplus; drosophila-melanogaster; differential expression; dermacentor-variabilis; ixodes-scapularis; resistant strain; mammalian-cells; salivary-glands; long dsrnas; cytochrome-p-450.
Subjects:Science > Zoology > Invertebrates > Insects
Animal culture > Cattle
Science > Biology > Genetics
Deposited On:06 Dec 2011 05:04
Last Modified:06 Dec 2011 05:04

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