title: Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation.
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.
authors.affiliation: Unidade de Experimentação Animal and Laboratório de Química e Função de Proteínas-Universidade Estadual Norte Fluminense-Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
authors.forename: Newton
authors.initials: N
authors.lastname: Ruiz
authors.affiliation: Laboratório Integrado de Bioquímica Hatisaburo Masuda-Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé (NUPEM/UFRJ), Macaé, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, Ilha do Fundão, RJ, Brazil.
authors.forename: Leonardo Araujo
authors.initials: LA
authors.lastname: de Abreu
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Luís Fernando
authors.initials: LF
authors.lastname: Parizi
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, United States of America.
authors.forename: Tae Kwon
authors.initials: TK
authors.lastname: Kim
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, United States of America.
authors.forename: Albert
authors.initials: A
authors.lastname: Mulenga
authors.affiliation: Departamento de Bioquímica-Instituto de Química, Universidade Federal do Rio de Janeiro, Ilha do Fundão, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, Ilha do Fundão, RJ, Brazil.
authors.forename: Gloria Regina Cardoso
authors.initials: GR
authors.lastname: Braz
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, Ilha do Fundão, RJ, Brazil.
authors.forename: Itabajara da Silva
authors.initials: Ida S
authors.lastname: Vaz
authors.affiliation: Unidade de Experimentação Animal and Laboratório de Química e Função de Proteínas-Universidade Estadual Norte Fluminense-Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, Ilha do Fundão, RJ, Brazil.
authors.forename: Carlos
authors.initials: C
authors.lastname: Logullo
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism; D018274:Electroporation; D005260:Female; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D038341:Glycogen Synthase Kinases / Q000235:genetics / Q000378:metabolism; D006536:Heptanes / Q000737:chemistry; D010063:Ovum / Q000737:chemistry / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D048494:Rhipicephalus / Q000235:genetics*; D012997:Solvents / Q000737:chemistry; D046509:Tissue Culture Techniques; D014885:Waxes / Q000737:chemistry
chemical_list: D060829:Arthropod Proteins; D006536:Heptanes; D012330:RNA, Double-Stranded; D012997:Solvents; D014885:Waxes; D038341:Glycogen Synthase Kinases; D051057:Proto-Oncogene Proteins c-akt