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.
Highlights
Ticks are haematophagous arthropods that target a wide range of terrestrial vertebrates
Consistency between replicates and duration of incubation with double stranded RNA (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
To investigate whether transfection reagents are required for tick cell lines and to determine transfection efficiency and toxicity, six different transfection reagents were tested on two tick cell lines, BDE/CTVM16 and IRE/CTVM19
Summary
Ticks are haematophagous arthropods that target a wide range of terrestrial vertebrates. While ticks can cause harm to their hosts directly through skin damage and blood loss, they transmit numerous bacteria, viruses and protozoa (Jongejan and Uilenberg 2004). Many of these are pathogenic for humans and/or domestic animals. Acaricide treatment and vaccination have been two main strategies for protection against ticks and tick-borne diseases. These measures have helped greatly, resistance to acaricide treatment is increasing, while vaccination is often impractical or insufficiently effective (George et al 2004; Willadsen 2006; Shkap et al 2007; de la Fuente et al 2007a; Latif and Hove 2011). Concurrent development of molecular biological tools and techniques such as RNA interference (RNAi), proteomics and transcriptomic analysis is greatly facilitating research into tick-host-pathogen interactions
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