Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation

• Published in: PloS one 2015-06, Vol.10 (6), p.e0130008-e0130008
• Main Authors: Ruiz, Newton, de Abreu, Leonardo Araujo, Parizi, Luís Fernando, Kim, Tae Kwon, Mulenga, Albert, Braz, Gloria Regina Cardoso, Vaz, Jr, Itabajara da Silva, Logullo, Carlos
• Format: Article
• Language: English
• Subjects: 4',6-Diamidino-2-phenylindole; AKT protein; Animals; Arachnids; Arthropod Proteins - genetics; Arthropod Proteins - metabolism; Boophilus; Bovidae; Cascades; Data processing; Deoxyribonucleic acid; DNA; Double-stranded RNA; Eggs; Electroporation; Embryogenesis; Embryonic development; Embryonic growth stage; Embryos; Energy metabolism; Female; Females; Fluorescence; Gene Expression; Gene Knockdown Techniques; Gene silencing; Genetic engineering; Glycogen; Glycogen synthase kinase 3; Glycogen Synthase Kinases - genetics; Glycogen Synthase Kinases - metabolism; Glycogen synthesis; Hatching; Heptanes; Heptanes - chemistry; Kinases; Metabolism; Molecular chains; Ovum - chemistry; Ovum - physiology; Primers; Proteins; Proto-Oncogene Proteins c-akt - genetics; Proto-Oncogene Proteins c-akt - metabolism; Reduction; Rhipicephalus; Rhipicephalus - genetics; Ribonucleic acid; RNA; RNA Interference; RNA, Double-Stranded - genetics; RNA-mediated interference; Solvents - chemistry; Tissue Culture Techniques; Tribolium castaneum; Vaccines; Veterinary colleges; Veterinary medicine; Waxes - chemistry
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0130008

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.