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
.jpg)
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/45178
Ver los metadatos del registro completo
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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 Técnicas2025-10-15T15:34:03Zoai: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:34982025-10-15 15:34:04.02CONICET 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 |
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info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
| eu_rights_str_mv |
openAccess |
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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 |
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CONICET Digital (CONICET) |
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CONICET Digital (CONICET) |
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Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
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dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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1846083469864599552 |
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13.22299 |