Sanscrainte Neil D, Waits Christy M, Geden Christopher J, Estep Alden S, Becnel James J
USDA/ARS Center for Medical, Agricultural, and Veterinary Entomology;
CMAVE Detachment, Navy Entomology Center of Excellence; Lovelace Respiratory Research Institute.
J Vis Exp. 2018 Nov 8(141). doi: 10.3791/58650.
Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds. Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts. This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.
用于诱导RNA干扰的合成双链RNA（dsRNA）可能具有剂量依赖性表型效应。如果使用非定量方法递送dsRNA，这些效应很难界定。准确递送已知量的核酸或其他化学物质对于衡量受试化合物的功效以及实现化合物之间可靠的比较至关重要。在此，我们提供了一种可重复的定量显微注射方案，可确保准确递送特定剂量的dsRNA，降低通常由注射损伤引起的死亡率。这些改进包括添加罗丹明B、使用刻度注射针以及借鉴Isoe和Collins的改进恢复方法。该方法能够计算剂量反应并便于化合物之间的比较。此方法的不同版本已成功应用于三类蚊子以及家蝇，以评估核糖体RNA转录本基因沉默导致的繁殖力降低情况。该方案提供了应对小型昆虫显微注射若干挑战的策略。总之，通过机械递送dsRNA并辅以视觉验证、确定有效的递送位置以及设置注射后恢复期，可确保准确给药并降低损伤死亡率。该方案还描述了一种用于统一测定对繁殖力影响的产卵生物测定法。
