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

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.

Fig. 1

Images of tick cells 24 h after addition of fluorescent siRNA (a–h) or dsRNA (i–l) in the presence or absence of a transfection reagent. a–d photomicrographs of BDE/CTVM16 cells taken at ×100 magnification with simultaneous normal transmitted light and UV reflected light to facilitate counting of fluorescent (green siRNA) and non-fluorescent cells. e–h confocal images of IRE/CTVM19 cells taken at ×630 magnification; cell nuclei are stained blue, while the siRNAs are green. i–l confocal images of IDE8 cells taken at ×630 magnification; cell nuclei are stained blue, while the long dsRNAs are green. a, e, i show untreated control cells; b, f, j show cells to which siRNA or dsRNA alone was added; c, g, k show cells to which siRNA or long dsRNA mixed with Lipofectamine 2000 was added; d, h, l show cells to which siRNA or long dsRNA mixed with Xtreme was added. (Color figure online)

Fig. 2

Silencing of a virus reporter protein in tick cells by siRNA treatment. a REE/CTVM28 cells were incubated with siRNA against RLuc in the presence or absence of Lipofectamine 2000 (Lipo) and then infected 24 h later with SFV expressing RLuc. Luciferase levels (y-axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Virus + siRNA + Lipo) and siRNA alone followed by SFV (Virus + siRNA only). The values shown are means of three replicate cultures and the error bars are standard deviations of the mean. * Significant decrease from the virus only control. b Nine tick cell lines were incubated with siRNA targeting RLuc in the presence of a transfection reagent and then infected 24 h later with SFV expressing RLuc. Luciferase levels (y-axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Lipo + siRNA + virus) and siRNA with Xtreme followed by SFV (Xtreme + siRNA + virus). The values shown are means of four replicate cultures and the error bars are standard deviations of the mean. The scale used for each y-axis reflects the range of luciferase levels generated in the particular tick cell line. * Significant decrease from the virus only control

Fig. 3

Silencing of a foreign virus reporter protein by long dsRNA treatment. Nine tick cell lines were incubated with long dsRNA targeting RLuc in the presence or absence of a transfection reagent and then infected 24 h later with SFV expressing RLuc. Luciferase levels (y-axis) were then measured 48 h later in cultures with SFV alone (Virus only), dsRNA followed by SFV (Virus + dsRNA), dsRNA with Lipofectamine 2000 followed by SFV (Lipo + dsRNA + virus) and dsRNA with Xtreme followed by SFV (Xtreme + dsRNA + virus). The values shown are means of four replicate cultures and the error bars are standard deviations of the mean. The scale used for each y-axis reflects the range of luciferase levels generated in the particular tick cell line. * Significant decrease from the virus only control

Fig. 4

Efficiency of subolesin mRNA transcript knockdown in IDE8 and ISE6 cells. IDE8 (upper) and ISE6 (lower) cells were transfected (with or without a transfection reagent) with dsRNA targeting subolesin or negative control dsRNA targeting eGFP and incubated for 96 h. Data for all replicates are included to illustrate the level of variability encountered between individual cultures. Total RNA was extracted and equal amounts of RNA were used to make cDNA. Transcripts were measured by real-time PCR and C_t values for subolesin mRNA were then normalised against tick β-actin mRNA for each replicate. Subolesin mRNA expression levels (y-axis) are shown in arbitrary units. –ve dsRNA targeting eGFP, dsRNA  only dsRNA targeting subolesin, lipo Lipofectamine 2000 + dsRNA targeting subolesin, x Xtreme + dsRNA targeting subolesin

Fig. 5

Efficiency of 2I1F6 mRNA transcript knockdown in IDE8 cells. IDE8 cells were transfected (without a transfection reagent) with dsRNA targeting 2I1F6 (2I1F6 dsRNA) or negative control dsRNA targeting eGFP (−ve dsRNA) and incubated for 24, 48, 72 or 96 h. Total RNA was extracted at each timepoint and equal amounts of RNA were used to make cDNA. Transcripts were measured by real-time PCR and C_t values for 2I1F6 mRNA were normalised against tick β-actin mRNA for each replicate. 2I1F6 mRNA expression levels (y-axis) are shown in arbitrary units. These values are means of four replicate cultures and the error bars are standard deviations from the mean. * significant difference between the −ve dsRNA and the 2I1F6 dsRNA