The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK.
Exp Appl Acarol. 2013 Mar;59(3):319-38. doi: 10.1007/s10493-012-9598-x. Epub 2012 Jul 7.
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.
RNA 干扰（RNAi）基因沉默是生物学许多领域的重要研究工具。为了有效地利用这项技术的力量来探索蜱的功能基因组学和蜱-微生物相互作用，需要建立优化的蜱细胞中 RNAi 介导的基因沉默的参数。从四个具有经济重要性的硬蜱属（硬蜱属、硬蜱属、硬蜱属和硬蜱属，包括亚种牛蜱属）中使用了十种细胞系，以检查包括小干扰 RNA（siRNA）、双链 RNA（dsRNA）、转染试剂和孵育时间在内的关键参数，以沉默病毒报告基因和内源性蜱基因。转染试剂对于 siRNA 的摄取是必需的，而长 dsRNA 则被大多数蜱细胞系摄取。在所测试的所有细胞系中，使用 siRNA 或 dsRNA 均可显著降低病毒报告蛋白的表达。最佳条件根据细胞系而有所不同。对于两种硬蜱属（硬蜱属和硬蜱属）的两个 Ixodes scapularis 细胞系，重复和与 dsRNA 孵育的时间之间的一致性得到了解决；IDE8 比 ISE6 更支持内源性基因 subolesin 的更一致和有效的沉默，并且在 48 小时内用 dsRNA 孵育 IDE8 细胞可实现内源性基因 2I1F6 的高度显著敲低。总之，这项研究表明，与 siRNA 相比，dsRNA 通常更有效地在蜱细胞系中进行基因沉默，但结果因细胞系而异，每个实验系统都需要确定最佳参数。
