Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition

Autores
Alessio, Ana Paula; Fili, Alejandro; Forcato, Diego Oscar; Olmos Nicotra, Maria Florencia; Alustiza, Fabrisio Eduardo; Rodriguez, Natalia Evelin; Sampaio, R. V.; Sangalli, J.; Bressan, F.; Fantinato-Neto, P.; Meirelles, F.; Owens, J.; Moisyadi, S.; Kues, W.A.; Bosch, Pablo
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117-8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19184-19189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey´s test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0±17.8 v. 100.0±16.1 and 2.8±0.8 respectively, n=4-7; P<0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development.
Fil: Alessio, Ana Paula. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Fili, Alejandro. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Forcato, Diego Oscar. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Olmos Nicotra, Maria Florencia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Alustiza, Fabrisio Eduardo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Rodriguez, Natalia Evelin. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Sampaio, R. V.. Universidade de Sao Paulo; Brasil
Fil: Sangalli, J.. Universidade de Sao Paulo; Brasil
Fil: Bressan, F.. Universidade de Sao Paulo; Brasil
Fil: Fantinato-Neto, P.. Universidade de Sao Paulo; Brasil
Fil: Meirelles, F.. Universidade de Sao Paulo; Brasil
Fil: Owens, J.. John A. Burns School Of Medicine; Estados Unidos
Fil: Moisyadi, S.. John A. Burns School Of Medicine; Estados Unidos
Fil: Kues, W.A.. Friedrich-Loeffler-Institut; Alemania
Fil: Bosch, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
Active Transgenesis
Transposon
Bovine
Nuclear Transfer
Piggybac
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/45178

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network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transpositionAlessio, Ana PaulaFili, AlejandroForcato, Diego OscarOlmos Nicotra, Maria FlorenciaAlustiza, Fabrisio EduardoRodriguez, Natalia EvelinSampaio, R. V.Sangalli, J.Bressan, F.Fantinato-Neto, P.Meirelles, F.Owens, J.Moisyadi, S.Kues, W.A.Bosch, PabloActive TransgenesisTransposonBovineNuclear TransferPiggybachttps://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117-8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19184-19189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey´s test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0±17.8 v. 100.0±16.1 and 2.8±0.8 respectively, n=4-7; P<0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development.Fil: Alessio, Ana Paula. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fili, Alejandro. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Forcato, Diego Oscar. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Olmos Nicotra, Maria Florencia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alustiza, Fabrisio Eduardo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Rodriguez, Natalia Evelin. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Sampaio, R. V.. Universidade de Sao Paulo; BrasilFil: Sangalli, J.. Universidade de Sao Paulo; BrasilFil: Bressan, F.. Universidade de Sao Paulo; BrasilFil: Fantinato-Neto, P.. Universidade de Sao Paulo; BrasilFil: Meirelles, F.. Universidade de Sao Paulo; BrasilFil: Owens, J.. John A. Burns School Of Medicine; Estados UnidosFil: Moisyadi, S.. John A. Burns School Of Medicine; Estados UnidosFil: Kues, W.A.. Friedrich-Loeffler-Institut; AlemaniaFil: Bosch, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaCsiro Publishing2014-12-04info: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/11336/45178Alessio, Ana Paula; Fili, Alejandro; Forcato, Diego Oscar; Olmos Nicotra, Maria Florencia; Alustiza, Fabrisio Eduardo; et al.; Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition; Csiro Publishing; Reproduction Fertility and Development; 27; 4-12-2014; 266-2671031-3613CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1071/RDv27n1Ab357info:eu-repo/semantics/altIdentifier/url/http://www.publish.csiro.au/rd/RDv27n1Ab357info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:36:29Zoai:ri.conicet.gov.ar:11336/45178instacron: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 10:36:29.768CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition
title Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition
spellingShingle Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition
Alessio, Ana Paula
Active Transgenesis
Transposon
Bovine
Nuclear Transfer
Piggybac
title_short Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition
title_full Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition
title_fullStr Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition
title_full_unstemmed Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition
title_sort Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition
dc.creator.none.fl_str_mv Alessio, Ana Paula
Fili, Alejandro
Forcato, Diego Oscar
Olmos Nicotra, Maria Florencia
Alustiza, Fabrisio Eduardo
Rodriguez, Natalia Evelin
Sampaio, R. V.
Sangalli, J.
Bressan, F.
Fantinato-Neto, P.
Meirelles, F.
Owens, J.
Moisyadi, S.
Kues, W.A.
Bosch, Pablo
author Alessio, Ana Paula
author_facet Alessio, Ana Paula
Fili, Alejandro
Forcato, Diego Oscar
Olmos Nicotra, Maria Florencia
Alustiza, Fabrisio Eduardo
Rodriguez, Natalia Evelin
Sampaio, R. V.
Sangalli, J.
Bressan, F.
Fantinato-Neto, P.
Meirelles, F.
Owens, J.
Moisyadi, S.
Kues, W.A.
Bosch, Pablo
author_role author
author2 Fili, Alejandro
Forcato, Diego Oscar
Olmos Nicotra, Maria Florencia
Alustiza, Fabrisio Eduardo
Rodriguez, Natalia Evelin
Sampaio, R. V.
Sangalli, J.
Bressan, F.
Fantinato-Neto, P.
Meirelles, F.
Owens, J.
Moisyadi, S.
Kues, W.A.
Bosch, Pablo
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Active Transgenesis
Transposon
Bovine
Nuclear Transfer
Piggybac
topic Active Transgenesis
Transposon
Bovine
Nuclear Transfer
Piggybac
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.4
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117-8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19184-19189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey´s test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0±17.8 v. 100.0±16.1 and 2.8±0.8 respectively, n=4-7; P<0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development.
Fil: Alessio, Ana Paula. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Fili, Alejandro. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Forcato, Diego Oscar. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Olmos Nicotra, Maria Florencia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Alustiza, Fabrisio Eduardo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Rodriguez, Natalia Evelin. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Sampaio, R. V.. Universidade de Sao Paulo; Brasil
Fil: Sangalli, J.. Universidade de Sao Paulo; Brasil
Fil: Bressan, F.. Universidade de Sao Paulo; Brasil
Fil: Fantinato-Neto, P.. Universidade de Sao Paulo; Brasil
Fil: Meirelles, F.. Universidade de Sao Paulo; Brasil
Fil: Owens, J.. John A. Burns School Of Medicine; Estados Unidos
Fil: Moisyadi, S.. John A. Burns School Of Medicine; Estados Unidos
Fil: Kues, W.A.. Friedrich-Loeffler-Institut; Alemania
Fil: Bosch, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117-8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19184-19189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey´s test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0±17.8 v. 100.0±16.1 and 2.8±0.8 respectively, n=4-7; P<0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development.
publishDate 2014
dc.date.none.fl_str_mv 2014-12-04
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/11336/45178
Alessio, Ana Paula; Fili, Alejandro; Forcato, Diego Oscar; Olmos Nicotra, Maria Florencia; Alustiza, Fabrisio Eduardo; et al.; Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition; Csiro Publishing; Reproduction Fertility and Development; 27; 4-12-2014; 266-267
1031-3613
CONICET Digital
CONICET
url http://hdl.handle.net/11336/45178
identifier_str_mv Alessio, Ana Paula; Fili, Alejandro; Forcato, Diego Oscar; Olmos Nicotra, Maria Florencia; Alustiza, Fabrisio Eduardo; et al.; Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition; Csiro Publishing; Reproduction Fertility and Development; 27; 4-12-2014; 266-267
1031-3613
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1071/RDv27n1Ab357
info:eu-repo/semantics/altIdentifier/url/http://www.publish.csiro.au/rd/RDv27n1Ab357
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Csiro Publishing
publisher.none.fl_str_mv Csiro Publishing
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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