Abstract
RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT) / Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis.
Highlights
Rhipicephalus (Boophilus) microplus is an ectoparasite that affects livestock, causing economic losses and transmits important cattle disease agents, like Babesia spp and Anaplasma marginalePLOS ONE | DOI:10.1371/journal.pone.0130008 June 19, 2015RNAi Silencing by Electroporation in Tick Eggs
In order to improve the knowledge about these metabolic processes, we have developed and evaluated a method to deliver double-stranded RNA (dsRNA) into R. microplus eggs
The RNA interference tool is widely used to study gene function. This method allows specific gene silencing through the synthesis and delivery of double-stranded RNA of cognate target gene
Summary
Rhipicephalus (Boophilus) microplus is an ectoparasite that affects livestock, causing economic losses and transmits important cattle disease agents, like Babesia spp and Anaplasma marginale. High fecundity allows thousands of larvae from a single female, being one of the most important factors that maintain elevated tick populations in environment. In this context, understanding the molecular basis of tick embryogenesis could be useful to aid the development of new control strategies. One drawback of conventional methods for double-stranded RNA (dsRNA) delivery is the potential of tick egg/embryo structure damage. We use the glucose metabolism related to cellular development [8] to evaluate the effect of silencing Protein Kinase B (AKT) and Glycogen Kinase Synthase (GSK) genes in tick embryos. We show that de-waxing using heptane prior to electroporation, improves gene silencing rates
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
