Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors

Autores
Bevacqua, Romina Jimena; Pereyra Bonnet, Federico Alberto; Olivera, Ramiro; Hiriart, María Inés; Sipowicz, Pablo; Fernández y Martín, Rafael; Radrizzani Helguera, Martin; Salamone, Daniel Felipe
Año de publicación
2012
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-β-actin enhancer promoter control] gene plasmid (50 ng/μL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 μm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.
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: Olivera, Ramiro. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina
Fil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Sipowicz, Pablo. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente. Laboratorio de Neuro y Citogenética Molecular; Argentina
Fil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Radrizzani Helguera, Martin. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente. Laboratorio de Neuro y Citogenética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; 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
6-Dimethylaminopurine
Dehydroleukodine
Transgenesis
Phosphorylated
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:11336/16349
Acceder
id CONICETDig_728ebb324ea172241e5040dbec27680a
oai_identifier_str oai:ri.conicet.gov.ar:11336/16349
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitorsBevacqua, Romina JimenaPereyra Bonnet, Federico AlbertoOlivera, RamiroHiriart, María InésSipowicz, PabloFernández y Martín, RafaelRadrizzani Helguera, MartinSalamone, Daniel Felipe6-DimethylaminopurineDehydroleukodineTransgenesisPhosphorylatedhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-β-actin enhancer promoter control] gene plasmid (50 ng/μL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 μm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.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: Olivera, Ramiro. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sipowicz, Pablo. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente. Laboratorio de Neuro y Citogenética Molecular; ArgentinaFil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Radrizzani Helguera, Martin. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente. Laboratorio de Neuro y Citogenética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; 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 Inc2012-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/16349Bevacqua, Romina Jimena; Pereyra Bonnet, Federico Alberto; Olivera, Ramiro; Hiriart, María Inés; Sipowicz, Pablo; et al.; Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors; Elsevier Science Inc; Theriogenology; 78; 1; 7-2012; 57-680093-691Xenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.theriogenology.2012.01.020info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0093691X12000556info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:35:18Zoai:ri.conicet.gov.ar:11336/16349instacron: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:34982025-09-29 09:35:18.906CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors
title Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors
spellingShingle Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors
Bevacqua, Romina Jimena
6-Dimethylaminopurine
Dehydroleukodine
Transgenesis
Phosphorylated
title_short Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors
title_full Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors
title_fullStr Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors
title_full_unstemmed Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors
title_sort Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors
dc.creator.none.fl_str_mv Bevacqua, Romina Jimena
Pereyra Bonnet, Federico Alberto
Olivera, Ramiro
Hiriart, María Inés
Sipowicz, Pablo
Fernández y Martín, Rafael
Radrizzani Helguera, Martin
Salamone, Daniel Felipe
author Bevacqua, Romina Jimena
author_facet Bevacqua, Romina Jimena
Pereyra Bonnet, Federico Alberto
Olivera, Ramiro
Hiriart, María Inés
Sipowicz, Pablo
Fernández y Martín, Rafael
Radrizzani Helguera, Martin
Salamone, Daniel Felipe
author_role author
author2 Pereyra Bonnet, Federico Alberto
Olivera, Ramiro
Hiriart, María Inés
Sipowicz, Pablo
Fernández y Martín, Rafael
Radrizzani Helguera, Martin
Salamone, Daniel Felipe
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv 6-Dimethylaminopurine
Dehydroleukodine
Transgenesis
Phosphorylated
topic 6-Dimethylaminopurine
Dehydroleukodine
Transgenesis
Phosphorylated
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-β-actin enhancer promoter control] gene plasmid (50 ng/μL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 μm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.
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: Olivera, Ramiro. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina
Fil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Sipowicz, Pablo. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente. Laboratorio de Neuro y Citogenética Molecular; Argentina
Fil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Radrizzani Helguera, Martin. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente. Laboratorio de Neuro y Citogenética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; 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 The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-β-actin enhancer promoter control] gene plasmid (50 ng/μL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 μm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.
publishDate 2012
dc.date.none.fl_str_mv 2012-07
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/11336/16349
Bevacqua, Romina Jimena; Pereyra Bonnet, Federico Alberto; Olivera, Ramiro; Hiriart, María Inés; Sipowicz, Pablo; et al.; Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors; Elsevier Science Inc; Theriogenology; 78; 1; 7-2012; 57-68
0093-691X
url http://hdl.handle.net/11336/16349
identifier_str_mv Bevacqua, Romina Jimena; Pereyra Bonnet, Federico Alberto; Olivera, Ramiro; Hiriart, María Inés; Sipowicz, Pablo; et al.; Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors; Elsevier Science Inc; Theriogenology; 78; 1; 7-2012; 57-68
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.2012.01.020
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0093691X12000556
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
application/pdf
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
_version_ 1844613098955079680
score 13.070432

Publicaciones similares