Barrero, R.

Bellgard, M.I.

Bruyeres, A.B.

Gondro, C.

Kurscheid, S.

Lew-Tabor, A.

Moolhuijzen, P.M.

Morgan, J.A.T.

Rodriguez-Valle, M.

Zhang, B.

English

Research Repository

TTP7: Ticks and Tick-borne pathogens International Conference. Zaragoza (Spain), 201182MICROARRAY  EVIDENCE  FOR  OFF  TARGET  EFFECTS IN  TICK  RNA INTERFERENCE EXPERIMENTS, AND  THE  LACK  OF  STRONG  CORRELATION  BETWEEN  DSRNA  AND ANTIBODY PHENOTYPES IN TICK IN VITRO TREATMENTS FOR VACCINE  CANDIDATE SCREENING.Corresponding Author: Ala Lew-Tabor, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Queensland, Brisbane, Australia. E-Mail: a.lewtabor@uq.edu.auAuthors: Lew-Tabor, A.E.1,2,3, Rodriguez Valle, M.1,2, Kurscheid, S.1,3, Barrero, R.3, Gondro, C.1,4, Moolhuijzen, P.M.1,3, Morgan, J.A.T.1,2, Bruyeres, A.B.1,5, Zhang, B.1,5, Bellgard, M.I.1,31Cooperative Research Centre for Beef Genetic Technologies, Armidale, NSW, Australia; 2Queensland Alliance for Agriculture  &  Food  Innovation,  The University  of  Queensland,  Qld, 4072, Australia;  3Centre for  Comparative Genomics, Murdoch University, Perth, Western Australia 6150, Australia;  4The Centre for Genetic Analysis  and Applications, University of New England, Armidale, NSW 2351, Australia; 5Department of Employment, Economic Development and Innovation, Locked Mail Bag No. 4, Moorooka, Brisbane, Qld 4105, Australia.KeywordsRNAi, microarray, vaccineKnowledge of  cattle tick  (Rhipicephalus  (Boophilus)  microplus; Acari:Ixodidae)  molecular  pathways  has  been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags available to date consist of ~50%  unassigned sequences  without predicted functions. The most common approach is  the application of RNA interference (RNAi) methods  to investigate gene function and to confirm potential of targeted genes  as vaccine antigens. This  has  been widely adopted despite lack  of knowledge of the tick  RNAi pathway, double-stranded RNA (dsRNA) uptake mechanisms, or host immune recognition of the targeted genes. A strong knockdown phenotype of adult female ticks had been previously observed using a 594 bp dsRNA targeting the cattle tick  homologue for the Drosophila Ubiquitin-63E gene leading to death or  nil and deformed eggs  if the female tick survived (Kurscheid et al. 2009). A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs  (14 x 50-mer probes  for 13,601 targets  of the total 13,643 ESTs) was  used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA. A total of 144 ESTs  including TC6372 (Ubiquitin-63E)  were down-regulated. The results  substantiated the knockdown phenotype  with  ESTs  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 ESTs  on the microarrays. Subsequent knockdown experiments  utilising  196  bp 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 real time PCR analysis confirmed differential expression of TC6372 and selected ESTs including OTE targets. We also compared female tick in vitro treatments using corresponding dsRNAs and antibodies  targeting 11  different  ESTs  known  to  be recognised  by  the  bovine  immune  system.  Only  two ESTs  demonstrated  a correlation between gene knockdown and antibody treatment resulting in death and poor egg production, while 7 demonstrated antibody effects  on survival but no effect following dsRNA treatment and 2 showed the converse with dsRNA effects on survival only. This is the first study addressing tick dsRNA delivery methods demonstrating the minimisation of predicted OTEs in shorter dsRNA treatments  (~100-200 bp) and also illustrating the need for caution when interpreting knockdown phenotypes. We also demonstrate that dsRNA knockdown phenotypes  will not always correlate to antibody or immune responses for the identification of putative vaccine antigens.Acknowledgements: Beef CRC, Queensland government.

Microarray evidence for off target effects in tick RNA interference experiments, and the lack of strong correlation between DSRNA and antibody phenotypes in tick In vitro treatments for vaccine candidate screening

http://researchrepository.murdoch.edu.au/13073/

Microarray evidence for off target effects in tick RNA interference experiments, and the lack of strong correlation between DSRNA and antibody phenotypes in tick In vitro treatments for vaccine candidate screening

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