Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation
Autor(a) principal: | |
---|---|
Data de Publicação: | 2015 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UFRGS |
Texto Completo: | http://hdl.handle.net/10183/224344 |
Resumo: | 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. |
id |
UFRGS-2_36ee556e0dcbcd5df812e5a190d85751 |
---|---|
oai_identifier_str |
oai:www.lume.ufrgs.br:10183/224344 |
network_acronym_str |
UFRGS-2 |
network_name_str |
Repositório Institucional da UFRGS |
repository_id_str |
|
spelling |
Ruiz, NewtonAbreu, Leonardo Araujo deParizi, Luis FernandoKim, Tae KwonMulenga, AlbertBraz, Glória Regina CardosoVaz Junior, Itabajara da SilvaLogullo, Carlos2021-07-23T04:40:19Z20151932-6203http://hdl.handle.net/10183/224344000969450RNA 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.application/pdfengPLoS ONE. San Francisco. Vol. 10 no. 6 (June 2015), e0130008, 13 p.Imunologia veterinaria : VacinasBiotecnologiaRhipicephalus microplusNon-invasive delivery of dsRNA into de-waxed tick eggs by electroporationEstrangeiroinfo:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFRGSinstname:Universidade Federal do Rio Grande do Sul (UFRGS)instacron:UFRGSTEXT000969450.pdf.txt000969450.pdf.txtExtracted Texttext/plain38496http://www.lume.ufrgs.br/bitstream/10183/224344/2/000969450.pdf.txt9817a60afeda2727a6d99ff8ad2899bdMD52ORIGINAL000969450.pdfTexto completo (inglês)application/pdf5113457http://www.lume.ufrgs.br/bitstream/10183/224344/1/000969450.pdf4f361f0b6c4e2f08e57eec0198516cc9MD5110183/2243442023-09-23 03:34:06.424354oai:www.lume.ufrgs.br:10183/224344Repositório de PublicaçõesPUBhttps://lume.ufrgs.br/oai/requestopendoar:2023-09-23T06:34:06Repositório Institucional da UFRGS - Universidade Federal do Rio Grande do Sul (UFRGS)false |
dc.title.pt_BR.fl_str_mv |
Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation |
title |
Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation |
spellingShingle |
Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation Ruiz, Newton Imunologia veterinaria : Vacinas Biotecnologia Rhipicephalus microplus |
title_short |
Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation |
title_full |
Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation |
title_fullStr |
Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation |
title_full_unstemmed |
Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation |
title_sort |
Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation |
author |
Ruiz, Newton |
author_facet |
Ruiz, Newton Abreu, Leonardo Araujo de Parizi, Luis Fernando Kim, Tae Kwon Mulenga, Albert Braz, Glória Regina Cardoso Vaz Junior, Itabajara da Silva Logullo, Carlos |
author_role |
author |
author2 |
Abreu, Leonardo Araujo de Parizi, Luis Fernando Kim, Tae Kwon Mulenga, Albert Braz, Glória Regina Cardoso Vaz Junior, Itabajara da Silva Logullo, Carlos |
author2_role |
author author author author author author author |
dc.contributor.author.fl_str_mv |
Ruiz, Newton Abreu, Leonardo Araujo de Parizi, Luis Fernando Kim, Tae Kwon Mulenga, Albert Braz, Glória Regina Cardoso Vaz Junior, Itabajara da Silva Logullo, Carlos |
dc.subject.por.fl_str_mv |
Imunologia veterinaria : Vacinas Biotecnologia Rhipicephalus microplus |
topic |
Imunologia veterinaria : Vacinas Biotecnologia Rhipicephalus microplus |
description |
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. |
publishDate |
2015 |
dc.date.issued.fl_str_mv |
2015 |
dc.date.accessioned.fl_str_mv |
2021-07-23T04:40:19Z |
dc.type.driver.fl_str_mv |
Estrangeiro info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10183/224344 |
dc.identifier.issn.pt_BR.fl_str_mv |
1932-6203 |
dc.identifier.nrb.pt_BR.fl_str_mv |
000969450 |
identifier_str_mv |
1932-6203 000969450 |
url |
http://hdl.handle.net/10183/224344 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.pt_BR.fl_str_mv |
PLoS ONE. San Francisco. Vol. 10 no. 6 (June 2015), e0130008, 13 p. |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFRGS instname:Universidade Federal do Rio Grande do Sul (UFRGS) instacron:UFRGS |
instname_str |
Universidade Federal do Rio Grande do Sul (UFRGS) |
instacron_str |
UFRGS |
institution |
UFRGS |
reponame_str |
Repositório Institucional da UFRGS |
collection |
Repositório Institucional da UFRGS |
bitstream.url.fl_str_mv |
http://www.lume.ufrgs.br/bitstream/10183/224344/2/000969450.pdf.txt http://www.lume.ufrgs.br/bitstream/10183/224344/1/000969450.pdf |
bitstream.checksum.fl_str_mv |
9817a60afeda2727a6d99ff8ad2899bd 4f361f0b6c4e2f08e57eec0198516cc9 |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional da UFRGS - Universidade Federal do Rio Grande do Sul (UFRGS) |
repository.mail.fl_str_mv |
|
_version_ |
1801225027482288128 |