Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Barry, G., Alberdi, P., Schnettler, E., Weisheit, S., Kohl, A. , Fazakerley, J.K. and Bell-Sakyi, L. (2013) Gene silencing in tick cell lines using small interfering or long double-stranded RNA. Experimental and Applied Acarology, 59(3), pp. 319-338. (doi:10.1007/s10493-012-9598-x)

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Abstract

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Schnettler, Dr Esther and Kohl, Professor Alain and Barry, Dr Gerald
Authors: Barry, G., Alberdi, P., Schnettler, E., Weisheit, S., Kohl, A., Fazakerley, J.K., and Bell-Sakyi, L.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Experimental and Applied Acarology
Publisher:Springer
ISSN:0168-8162
ISSN (Online):1572-9702
Published Online:07 July 2012
Copyright Holders:Copyright © 2012 The Authors
First Published:First published in Expermental and Applied Acarology 59(3):319-338
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
531961POSTICK - Post-graduate training network for capacity building to control ticks and tick-borne diseasesBrian ShielsEuropean Commission (EC)238511 POSTICKIII - PARASITOLOGY