Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus
Autor(a) principal: | |
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Data de Publicação: | 2009 |
Outros Autores: | , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da FURG (RI FURG) |
Texto Completo: | http://repositorio.furg.br/handle/1/3673 http://dx.doi.org/10.1016/j.theriogenology.2008.08.019 |
Resumo: | Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/106 cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa;controlling this activity improved the efficiency of SMGT in fish. |
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Lanes, Carlos Frederico CecconSampaio, Luís André Nassr deMarins, Luis Fernando Fernandes2013-08-14T19:28:50Z2013-08-14T19:28:50Z2009LANES, Carlos Frederico Ceccon; SAMPAIO, Luís André Nassr de; MARINS, Luis Fernando Fernandes. Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus. Theriogenology, v. 71, n. 3, p. 525-533, 2009. Disponível em: <http://ac.els-cdn.com/S0093691X08006158/1-s2.0-S0093691X08006158-main.pdf?_tid=21ba7fc6-2b79-11e2-b805-00000aacb360&acdnat=1352581183_717767629af657dce6b322b3297c704d>. Acesso em: 10 nov. 2012.http://repositorio.furg.br/handle/1/3673http://dx.doi.org/10.1016/j.theriogenology.2008.08.019Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/106 cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa;controlling this activity improved the efficiency of SMGT in fish.engDNaseSpermatozoaDNA uptakeSMGTParalichthys orbignyanusEvaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanusinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da FURG (RI FURG)instname:Universidade Federal do Rio Grande (FURG)instacron:FURGORIGINALEvaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus.pdfEvaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus.pdfapplication/pdf590842https://repositorio.furg.br/bitstream/1/3673/1/Evaluation%20of%20DNase%20activity%20in%20seminal%20plasma%20and%20uptake%20of%20exogenous%20DNA%20by%20spermatozoa%20of%20the%20Brazilian%20flounder%20Paralichthys%20orbignyanus.pdf3c672058cce1bc85f00ba0f3e9c1550eMD51open accessLICENSElicense.txtlicense.txttext/plain; charset=utf-81724https://repositorio.furg.br/bitstream/1/3673/2/license.txt5b92b9704b4f13242d70e45ddef35a68MD52open access1/36732013-08-14 16:28:50.527open accessoai:repositorio.furg.br:1/3673w4kgbmVjZXNzw6FyaW8gY29uY29yZGFyIGNvbSBhIGxpY2Vuw6dhIGRlIGRpc3RyaWJ1acOnw6NvIG7Do28tZXhjbHVzaXZhLAphbnRlcyBxdWUgbyBkb2N1bWVudG8gcG9zc2EgYXBhcmVjZXIgbm8gUmVwb3NpdMOzcmlvLiBQb3IgZmF2b3IsIGxlaWEgYQpsaWNlbsOnYSBhdGVudGFtZW50ZS4gQ2FzbyBuZWNlc3NpdGUgZGUgYWxndW0gZXNjbGFyZWNpbWVudG8gZW50cmUgZW0KY29udGF0byBhdHJhdsOpcyBkZTogcmVwb3NpdG9yaW9AZnVyZy5iciBvdSAweHggNTMgMzIzMy02NzA2LgoKTElDRU7Dh0EgREUgRElTVFJJQlVJw4fDg08gTsODTy1FWENMVVNJVkEKCkFvIGFzc2luYXIgZSBlbnRyZWdhciBlc3RhIGxpY2Vuw6dhLCBvL2EgU3IuL1NyYS4gKGF1dG9yIG91IGRldGVudG9yIGRvcyBkaXJlaXRvcyBkZSBhdXRvcik6CgphKSBDb25jZWRlIMOgIFVuaXZlcnNpZGFkZSBGZWRlcmFsIGRlIFJpbyBHcmFuZGUgLSAgRlVSRyBvIGRpcmVpdG8gbsOjby1leGNsdXNpdm8gZGUgcmVwcm9kdXppciwgY29udmVydGVyIChjb21vIGRlZmluaWRvIGFiYWl4byksIGNvbXVuaWNhciBlL291IGRpc3RyaWJ1aXIgbyBkb2N1bWVudG8gZW50cmVndWUgKGluY2x1aW5kbyBvIHJlc3Vtby9hYnN0cmFjdCkgZW0KZm9ybWF0byBkaWdpdGFsIG91IGltcHJlc3NvIGUgZW0gcXVhbHF1ZXIgbWVpby4KCmIpIERlY2xhcmEgcXVlIG8gZG9jdW1lbnRvIGVudHJlZ3VlIMOpIHNldSB0cmFiYWxobyBvcmlnaW5hbCwgZSBxdWUKZGV0w6ltIG8gZGlyZWl0byBkZSBjb25jZWRlciBvcyBkaXJlaXRvcyBjb250aWRvcyBuZXN0YSBsaWNlbsOnYS4gRGVjbGFyYSB0YW1iw6ltIHF1ZSBhIGVudHJlZ2EgZG8gZG9jdW1lbnRvIG7Do28gaW5mcmluZ2UsIHRhbnRvIHF1YW50byBsaGUgw6kgcG9zc8OtdmVsIHNhYmVyLCBvcyBkaXJlaXRvcyBkZSBxdWFscXVlciBvdXRyYSBwZXNzb2Egb3UgZW50aWRhZGUuCgpjKSBTZSBvIGRvY3VtZW50byBlbnRyZWd1ZSBjb250w6ltIG1hdGVyaWFsIGRvIHF1YWwgbsOjbyBkZXTDqW0gb3MKZGlyZWl0b3MgZGUgYXV0b3IsIGRlY2xhcmEgcXVlIG9idGV2ZSBhdXRvcml6YcOnw6NvIGRvIGRldGVudG9yIGRvcwpkaXJlaXRvcyBkZSBhdXRvciBwYXJhIGNvbmNlZGVyIMOgIEZVUkcgb3MgZGlyZWl0b3MgcmVxdWVyaWRvcyBwb3IgZXN0YSBsaWNlbsOnYSwgZSBxdWUgZXNzZSBtYXRlcmlhbCBjdWpvcyBkaXJlaXRvcyBzw6NvIGRlIHRlcmNlaXJvcyBlc3TDoSBjbGFyYW1lbnRlIGlkZW50aWZpY2FkbyBlIHJlY29uaGVjaWRvIG5vIHRleHRvIG91IGNvbnRlw7pkbyBkbyBkb2N1bWVudG8gZW50cmVndWUuCgpTZSBvIGRvY3VtZW50byBlbnRyZWd1ZSDDqSBiYXNlYWRvIGVtIHRyYWJhbGhvIGZpbmFuY2lhZG8gb3UgYXBvaWFkbwpwb3Igb3V0cmEgaW5zdGl0dWnDp8OjbyBxdWUgbsOjbyBhIEZVUkcsIGRlY2xhcmEgcXVlIGN1bXByaXUgcXVhaXNxdWVyIG9icmlnYcOnw7VlcyBleGlnaWRhcyBwZWxvIHJlc3BlY3Rpdm8gY29udHJhdG8gb3UgYWNvcmRvLgoKQSBGVVJHIGlkZW50aWZpY2Fyw6EgY2xhcmFtZW50ZSBvKHMpIHNldSAocykgbm9tZSAocykgY29tbyBvIChzKSBhdXRvciAoZXMpIG91IGRldGVudG9yIChlcykgZG9zIGRpcmVpdG9zIGRvIGRvY3VtZW50byBlbnRyZWd1ZSwgZSBuw6NvIGZhcsOhIHF1YWxxdWVyIGFsdGVyYcOnw6NvLCBwYXJhIGFsw6ltIGRhcyBwZXJtaXRpZGFzIHBvciBlc3RhIGxpY2Vuw6dhLgo=Repositório InstitucionalPUBhttps://repositorio.furg.br/oai/request || http://200.19.254.174/oai/requestopendoar:2013-08-14T19:28:50Repositório Institucional da FURG (RI FURG) - Universidade Federal do Rio Grande (FURG)false |
dc.title.pt_BR.fl_str_mv |
Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus |
title |
Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus |
spellingShingle |
Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus Lanes, Carlos Frederico Ceccon DNase Spermatozoa DNA uptake SMGT Paralichthys orbignyanus |
title_short |
Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus |
title_full |
Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus |
title_fullStr |
Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus |
title_full_unstemmed |
Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus |
title_sort |
Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus |
author |
Lanes, Carlos Frederico Ceccon |
author_facet |
Lanes, Carlos Frederico Ceccon Sampaio, Luís André Nassr de Marins, Luis Fernando Fernandes |
author_role |
author |
author2 |
Sampaio, Luís André Nassr de Marins, Luis Fernando Fernandes |
author2_role |
author author |
dc.contributor.author.fl_str_mv |
Lanes, Carlos Frederico Ceccon Sampaio, Luís André Nassr de Marins, Luis Fernando Fernandes |
dc.subject.por.fl_str_mv |
DNase Spermatozoa DNA uptake SMGT Paralichthys orbignyanus |
topic |
DNase Spermatozoa DNA uptake SMGT Paralichthys orbignyanus |
description |
Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/106 cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa;controlling this activity improved the efficiency of SMGT in fish. |
publishDate |
2009 |
dc.date.issued.fl_str_mv |
2009 |
dc.date.accessioned.fl_str_mv |
2013-08-14T19:28:50Z |
dc.date.available.fl_str_mv |
2013-08-14T19:28:50Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
LANES, Carlos Frederico Ceccon; SAMPAIO, Luís André Nassr de; MARINS, Luis Fernando Fernandes. Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus. Theriogenology, v. 71, n. 3, p. 525-533, 2009. Disponível em: <http://ac.els-cdn.com/S0093691X08006158/1-s2.0-S0093691X08006158-main.pdf?_tid=21ba7fc6-2b79-11e2-b805-00000aacb360&acdnat=1352581183_717767629af657dce6b322b3297c704d>. Acesso em: 10 nov. 2012. |
dc.identifier.uri.fl_str_mv |
http://repositorio.furg.br/handle/1/3673 |
dc.identifier.doi.pt_BR.fl_str_mv |
http://dx.doi.org/10.1016/j.theriogenology.2008.08.019 |
identifier_str_mv |
LANES, Carlos Frederico Ceccon; SAMPAIO, Luís André Nassr de; MARINS, Luis Fernando Fernandes. Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus. Theriogenology, v. 71, n. 3, p. 525-533, 2009. Disponível em: <http://ac.els-cdn.com/S0093691X08006158/1-s2.0-S0093691X08006158-main.pdf?_tid=21ba7fc6-2b79-11e2-b805-00000aacb360&acdnat=1352581183_717767629af657dce6b322b3297c704d>. Acesso em: 10 nov. 2012. |
url |
http://repositorio.furg.br/handle/1/3673 http://dx.doi.org/10.1016/j.theriogenology.2008.08.019 |
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eng |
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FURG |
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Repositório Institucional da FURG (RI FURG) |
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