High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation

Autores
Bevacqua, Romina Jimena; Pereyra Bonnet, Federico Alberto; Fernandez Martin, Rafael; Salamone, Daniel Felipe
Año de publicación
2010
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.
Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Pereyra Bonnet, Federico Alberto. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Fernandez Martin, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
Genetic Modification
Embryo Cultured
Explants Cells
Intracytoplasmic Sperm Injection
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/16191
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/16191
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activationBevacqua, Romina JimenaPereyra Bonnet, Federico AlbertoFernandez Martin, RafaelSalamone, Daniel FelipeGenetic ModificationEmbryo CulturedExplants CellsIntracytoplasmic Sperm Injectionhttps://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez Martin, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaElsevier Science Inc2010-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/16191Bevacqua, Romina Jimena; Pereyra Bonnet, Federico Alberto; Fernandez Martin, Rafael; Salamone, Daniel Felipe; High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation; Elsevier Science Inc; Theriogenology; 74; 6; 10-2010; 922-9310093-691Xenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.theriogenology.2010.04.017info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0093691X10002244info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T12:04:13Zoai:ri.conicet.gov.ar:11336/16191instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 12:04:13.671CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation
title High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation
spellingShingle High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation
Bevacqua, Romina Jimena
Genetic Modification
Embryo Cultured
Explants Cells
Intracytoplasmic Sperm Injection
title_short High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation
title_full High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation
title_fullStr High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation
title_full_unstemmed High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation
title_sort High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation
dc.creator.none.fl_str_mv Bevacqua, Romina Jimena
Pereyra Bonnet, Federico Alberto
Fernandez Martin, Rafael
Salamone, Daniel Felipe
author Bevacqua, Romina Jimena
author_facet Bevacqua, Romina Jimena
Pereyra Bonnet, Federico Alberto
Fernandez Martin, Rafael
Salamone, Daniel Felipe
author_role author
author2 Pereyra Bonnet, Federico Alberto
Fernandez Martin, Rafael
Salamone, Daniel Felipe
author2_role author
author
author
dc.subject.none.fl_str_mv Genetic Modification
Embryo Cultured
Explants Cells
Intracytoplasmic Sperm Injection
topic Genetic Modification
Embryo Cultured
Explants Cells
Intracytoplasmic Sperm Injection
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.4
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.
Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Pereyra Bonnet, Federico Alberto. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Fernandez Martin, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.
publishDate 2010
dc.date.none.fl_str_mv 2010-10
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/16191
Bevacqua, Romina Jimena; Pereyra Bonnet, Federico Alberto; Fernandez Martin, Rafael; Salamone, Daniel Felipe; High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation; Elsevier Science Inc; Theriogenology; 74; 6; 10-2010; 922-931
0093-691X
url http://hdl.handle.net/11336/16191
identifier_str_mv Bevacqua, Romina Jimena; Pereyra Bonnet, Federico Alberto; Fernandez Martin, Rafael; Salamone, Daniel Felipe; High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation; Elsevier Science Inc; Theriogenology; 74; 6; 10-2010; 922-931
0093-691X
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.theriogenology.2010.04.017
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0093691X10002244
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science Inc
publisher.none.fl_str_mv Elsevier Science Inc
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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