Please use this identifier to cite or link to this item: 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)orcid ; 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
Appears in Collections:Journal Article

Files in This Item:
2 files
File Description SizeFormat 
Show full item record

SCOPUSTM   
Citations

29
checked on Feb 17, 2024

Page view(s)

938
checked on Jun 11, 2023
Google Media

Google ScholarTM

Check

Altmetric


Items in Research UNE are protected by copyright, with all rights reserved, unless otherwise indicated.