Sleeping beauty transgenesis in cattle

Autores
Garrels, Wiebke; Talluri, Thirumala Rao; Bevaqua, Romina; Alessio, Ana Paula; Fili, Alejandro; Forcato, Diego Oscar; Rodriguez, Nancy; Olmos Nicotra, Maria Florencia; Ivics, Zálthan; Salamone, Daniel Felipe; Bosch, Pablo; Kues, Wilfried
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the Sleeping Beauty (SB) transposon system was assessed for stable gene transfer into the cattle genome. The transposon plasmids encoded a ubiquitously active CAGGS promoter-driven Venus reporter and a lens-specific α A-crystallin promoter driven tdTomato fluorophore, respectively. The helper plasmid carried the hyperactive SB100x transposase variant. In total, 50 in vitro-derived zygotes were co-injected (Garrels et al. 2011 PLoS ONE 6; Ivics et al. 2014 Nat. Protoc. 9) and cultured up to blastocyst stage (Day 8). Two blastocysts were Venus-positive and were transferred to synchronized heifers, resulting in one pregnancy. The resulting calf was normally developed and vital; however, it died shortly after cesarean section due to spontaneous bleeding from an undetected aneurism. Phenotypic analysis suggested that the calf was indeed double-transgenic, showing widespread expression of Venus and lens-specific expression of tdTomato. Genotyping and molecular analyses confirmed the integration of both reporter transposons and the faithful promoter-dependent expression patterns. Subdermal tissue of an ear biopsy was used to culture fibroblasts, which were employed in somatic cell nuclear transfer experiments. In total, 39 embryos were reconstructed, of which 34 underwent cleavage, and at the end of culture 12 morulas and 12 blastocysts were obtained. Ten of the blastocysts were Venus positive, and embryo transfer of Venus-positive blastocysts is planned. In summary, we showed that the cytoplasmic injection of SB components is a highly efficient method for transgenesis in cattle. Due to the modular composition of SB plasmids, even double transgenic cattle can be generated in a one-step procedure. Importantly, the SB-catalyzed integration seems to favour transcriptionally permissive loci in the genome, resulting in faithful and robust promoter-dependent expression of the transgenes. The transposon constructs carry heterospecific loxP sites, which will be instrumental for targeted insertion of functional transgenes by Cre recombinase-mediated cassette exchange.
Fil: Garrels, Wiebke. Institut für Nutztiergenetik; Alemania. Medical School Hannover; Alemania
Fil: Talluri, Thirumala Rao. Institut für Nutztiergenetik; Alemania
Fil: Bevaqua, Romina. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina
Fil: Alessio, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Fili, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Forcato, Diego Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Rodriguez, Nancy. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Olmos Nicotra, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Ivics, Zálthan. Paul-Ehrlich-Institute; Alemania
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina
Fil: Bosch, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Kues, Wilfried. Institut für Nutztiergenetik; Alemania
Materia
Active Trasngenesis
Transposon
Sleeping Beauty
Livestock
Nuclear Transfer
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:11336/45176
Acceder
id CONICETDig_557f8a616d027ced0a180cf683cbdac5
oai_identifier_str oai:ri.conicet.gov.ar:11336/45176
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Sleeping beauty transgenesis in cattleGarrels, WiebkeTalluri, Thirumala RaoBevaqua, RominaAlessio, Ana PaulaFili, AlejandroForcato, Diego OscarRodriguez, NancyOlmos Nicotra, Maria FlorenciaIvics, ZálthanSalamone, Daniel FelipeBosch, PabloKues, WilfriedActive TrasngenesisTransposonSleeping BeautyLivestockNuclear Transferhttps://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the Sleeping Beauty (SB) transposon system was assessed for stable gene transfer into the cattle genome. The transposon plasmids encoded a ubiquitously active CAGGS promoter-driven Venus reporter and a lens-specific α A-crystallin promoter driven tdTomato fluorophore, respectively. The helper plasmid carried the hyperactive SB100x transposase variant. In total, 50 in vitro-derived zygotes were co-injected (Garrels et al. 2011 PLoS ONE 6; Ivics et al. 2014 Nat. Protoc. 9) and cultured up to blastocyst stage (Day 8). Two blastocysts were Venus-positive and were transferred to synchronized heifers, resulting in one pregnancy. The resulting calf was normally developed and vital; however, it died shortly after cesarean section due to spontaneous bleeding from an undetected aneurism. Phenotypic analysis suggested that the calf was indeed double-transgenic, showing widespread expression of Venus and lens-specific expression of tdTomato. Genotyping and molecular analyses confirmed the integration of both reporter transposons and the faithful promoter-dependent expression patterns. Subdermal tissue of an ear biopsy was used to culture fibroblasts, which were employed in somatic cell nuclear transfer experiments. In total, 39 embryos were reconstructed, of which 34 underwent cleavage, and at the end of culture 12 morulas and 12 blastocysts were obtained. Ten of the blastocysts were Venus positive, and embryo transfer of Venus-positive blastocysts is planned. In summary, we showed that the cytoplasmic injection of SB components is a highly efficient method for transgenesis in cattle. Due to the modular composition of SB plasmids, even double transgenic cattle can be generated in a one-step procedure. Importantly, the SB-catalyzed integration seems to favour transcriptionally permissive loci in the genome, resulting in faithful and robust promoter-dependent expression of the transgenes. The transposon constructs carry heterospecific loxP sites, which will be instrumental for targeted insertion of functional transgenes by Cre recombinase-mediated cassette exchange.Fil: Garrels, Wiebke. Institut für Nutztiergenetik; Alemania. Medical School Hannover; AlemaniaFil: Talluri, Thirumala Rao. Institut für Nutztiergenetik; AlemaniaFil: Bevaqua, Romina. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; ArgentinaFil: Alessio, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Fili, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Forcato, Diego Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Rodriguez, Nancy. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Olmos Nicotra, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Ivics, Zálthan. Paul-Ehrlich-Institute; AlemaniaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; ArgentinaFil: Bosch, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Kues, Wilfried. Institut für Nutztiergenetik; AlemaniaCsiro 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/11336/45176Garrels, Wiebke; Talluri, Thirumala Rao; Bevaqua, Romina; Alessio, Ana Paula; Fili, Alejandro; et al.; Sleeping beauty transgenesis in cattle; Csiro Publishing; Reproduction Fertility and Development; 27; 4-12-2014; 266-2661031-36131448-5990CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1071/RDv27n1Ab356info:eu-repo/semantics/altIdentifier/url/http://www.publish.csiro.au/rd/RDv27n1Ab356info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T12:14:52Zoai:ri.conicet.gov.ar:11336/45176instacron: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 12:14:52.725CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Sleeping beauty transgenesis in cattle
title Sleeping beauty transgenesis in cattle
spellingShingle Sleeping beauty transgenesis in cattle
Garrels, Wiebke
Active Trasngenesis
Transposon
Sleeping Beauty
Livestock
Nuclear Transfer
title_short Sleeping beauty transgenesis in cattle
title_full Sleeping beauty transgenesis in cattle
title_fullStr Sleeping beauty transgenesis in cattle
title_full_unstemmed Sleeping beauty transgenesis in cattle
title_sort Sleeping beauty transgenesis in cattle
dc.creator.none.fl_str_mv Garrels, Wiebke
Talluri, Thirumala Rao
Bevaqua, Romina
Alessio, Ana Paula
Fili, Alejandro
Forcato, Diego Oscar
Rodriguez, Nancy
Olmos Nicotra, Maria Florencia
Ivics, Zálthan
Salamone, Daniel Felipe
Bosch, Pablo
Kues, Wilfried
author Garrels, Wiebke
author_facet Garrels, Wiebke
Talluri, Thirumala Rao
Bevaqua, Romina
Alessio, Ana Paula
Fili, Alejandro
Forcato, Diego Oscar
Rodriguez, Nancy
Olmos Nicotra, Maria Florencia
Ivics, Zálthan
Salamone, Daniel Felipe
Bosch, Pablo
Kues, Wilfried
author_role author
author2 Talluri, Thirumala Rao
Bevaqua, Romina
Alessio, Ana Paula
Fili, Alejandro
Forcato, Diego Oscar
Rodriguez, Nancy
Olmos Nicotra, Maria Florencia
Ivics, Zálthan
Salamone, Daniel Felipe
Bosch, Pablo
Kues, Wilfried
author2_role author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Active Trasngenesis
Transposon
Sleeping Beauty
Livestock
Nuclear Transfer
topic Active Trasngenesis
Transposon
Sleeping Beauty
Livestock
Nuclear Transfer
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 modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the Sleeping Beauty (SB) transposon system was assessed for stable gene transfer into the cattle genome. The transposon plasmids encoded a ubiquitously active CAGGS promoter-driven Venus reporter and a lens-specific α A-crystallin promoter driven tdTomato fluorophore, respectively. The helper plasmid carried the hyperactive SB100x transposase variant. In total, 50 in vitro-derived zygotes were co-injected (Garrels et al. 2011 PLoS ONE 6; Ivics et al. 2014 Nat. Protoc. 9) and cultured up to blastocyst stage (Day 8). Two blastocysts were Venus-positive and were transferred to synchronized heifers, resulting in one pregnancy. The resulting calf was normally developed and vital; however, it died shortly after cesarean section due to spontaneous bleeding from an undetected aneurism. Phenotypic analysis suggested that the calf was indeed double-transgenic, showing widespread expression of Venus and lens-specific expression of tdTomato. Genotyping and molecular analyses confirmed the integration of both reporter transposons and the faithful promoter-dependent expression patterns. Subdermal tissue of an ear biopsy was used to culture fibroblasts, which were employed in somatic cell nuclear transfer experiments. In total, 39 embryos were reconstructed, of which 34 underwent cleavage, and at the end of culture 12 morulas and 12 blastocysts were obtained. Ten of the blastocysts were Venus positive, and embryo transfer of Venus-positive blastocysts is planned. In summary, we showed that the cytoplasmic injection of SB components is a highly efficient method for transgenesis in cattle. Due to the modular composition of SB plasmids, even double transgenic cattle can be generated in a one-step procedure. Importantly, the SB-catalyzed integration seems to favour transcriptionally permissive loci in the genome, resulting in faithful and robust promoter-dependent expression of the transgenes. The transposon constructs carry heterospecific loxP sites, which will be instrumental for targeted insertion of functional transgenes by Cre recombinase-mediated cassette exchange.
Fil: Garrels, Wiebke. Institut für Nutztiergenetik; Alemania. Medical School Hannover; Alemania
Fil: Talluri, Thirumala Rao. Institut für Nutztiergenetik; Alemania
Fil: Bevaqua, Romina. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina
Fil: Alessio, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Fili, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Forcato, Diego Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Rodriguez, Nancy. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Olmos Nicotra, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Ivics, Zálthan. Paul-Ehrlich-Institute; Alemania
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina
Fil: Bosch, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
Fil: Kues, Wilfried. Institut für Nutztiergenetik; Alemania
description Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the Sleeping Beauty (SB) transposon system was assessed for stable gene transfer into the cattle genome. The transposon plasmids encoded a ubiquitously active CAGGS promoter-driven Venus reporter and a lens-specific α A-crystallin promoter driven tdTomato fluorophore, respectively. The helper plasmid carried the hyperactive SB100x transposase variant. In total, 50 in vitro-derived zygotes were co-injected (Garrels et al. 2011 PLoS ONE 6; Ivics et al. 2014 Nat. Protoc. 9) and cultured up to blastocyst stage (Day 8). Two blastocysts were Venus-positive and were transferred to synchronized heifers, resulting in one pregnancy. The resulting calf was normally developed and vital; however, it died shortly after cesarean section due to spontaneous bleeding from an undetected aneurism. Phenotypic analysis suggested that the calf was indeed double-transgenic, showing widespread expression of Venus and lens-specific expression of tdTomato. Genotyping and molecular analyses confirmed the integration of both reporter transposons and the faithful promoter-dependent expression patterns. Subdermal tissue of an ear biopsy was used to culture fibroblasts, which were employed in somatic cell nuclear transfer experiments. In total, 39 embryos were reconstructed, of which 34 underwent cleavage, and at the end of culture 12 morulas and 12 blastocysts were obtained. Ten of the blastocysts were Venus positive, and embryo transfer of Venus-positive blastocysts is planned. In summary, we showed that the cytoplasmic injection of SB components is a highly efficient method for transgenesis in cattle. Due to the modular composition of SB plasmids, even double transgenic cattle can be generated in a one-step procedure. Importantly, the SB-catalyzed integration seems to favour transcriptionally permissive loci in the genome, resulting in faithful and robust promoter-dependent expression of the transgenes. The transposon constructs carry heterospecific loxP sites, which will be instrumental for targeted insertion of functional transgenes by Cre recombinase-mediated cassette exchange.
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/11336/45176
Garrels, Wiebke; Talluri, Thirumala Rao; Bevaqua, Romina; Alessio, Ana Paula; Fili, Alejandro; et al.; Sleeping beauty transgenesis in cattle; Csiro Publishing; Reproduction Fertility and Development; 27; 4-12-2014; 266-266
1031-3613
1448-5990
CONICET Digital
CONICET
url http://hdl.handle.net/11336/45176
identifier_str_mv Garrels, Wiebke; Talluri, Thirumala Rao; Bevaqua, Romina; Alessio, Ana Paula; Fili, Alejandro; et al.; Sleeping beauty transgenesis in cattle; Csiro Publishing; Reproduction Fertility and Development; 27; 4-12-2014; 266-266
1031-3613
1448-5990
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/RDv27n1Ab356
info:eu-repo/semantics/altIdentifier/url/http://www.publish.csiro.au/rd/RDv27n1Ab356
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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score 12.982451

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