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dc.contributor.author
Garrels, Wiebke
dc.contributor.author
Talluri, Thirumala Rao
dc.contributor.author
Bevaqua, Romina
dc.contributor.author
Alessio, Ana Paula
dc.contributor.author
Fili, Alejandro
dc.contributor.author
Forcato, Diego Oscar
dc.contributor.author
Rodriguez, Nancy
dc.contributor.author
Olmos Nicotra, Maria Florencia
dc.contributor.author
Ivics, Zálthan
dc.contributor.author
Salamone, Daniel Felipe
dc.contributor.author
Bosch, Pablo
dc.contributor.author
Kues, Wilfried
dc.date.available
2018-05-15T13:37:47Z
dc.date.issued
2014-12-04
dc.identifier.citation
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
dc.identifier.issn
1031-3613
dc.identifier.uri
http://hdl.handle.net/11336/45176
dc.description.abstract
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.
dc.format
application/pdf
dc.language.iso
eng
dc.publisher
Csiro Publishing
dc.rights
info:eu-repo/semantics/restrictedAccess
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.subject
Active Trasngenesis
dc.subject
Transposon
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Sleeping Beauty
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Livestock
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Nuclear Transfer
dc.subject.classification
Otras Biotecnología Agropecuaria
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Biotecnología Agropecuaria
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CIENCIAS AGRÍCOLAS
dc.title
Sleeping beauty transgenesis in cattle
dc.type
info:eu-repo/semantics/article
dc.type
info:ar-repo/semantics/artículo
dc.type
info:eu-repo/semantics/publishedVersion
dc.date.updated
2018-03-02T13:58:51Z
dc.identifier.eissn
1448-5990
dc.journal.volume
27
dc.journal.pagination
266-266
dc.journal.pais
Australia
dc.journal.ciudad
Collingwood
dc.description.fil
Fil: Garrels, Wiebke. Institut für Nutztiergenetik; Alemania. Medical School Hannover; Alemania
dc.description.fil
Fil: Talluri, Thirumala Rao. Institut für Nutztiergenetik; Alemania
dc.description.fil
Fil: Bevaqua, Romina. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina
dc.description.fil
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
dc.description.fil
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
dc.description.fil
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
dc.description.fil
Fil: Rodriguez, Nancy. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina
dc.description.fil
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
dc.description.fil
Fil: Ivics, Zálthan. Paul-Ehrlich-Institute; Alemania
dc.description.fil
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina
dc.description.fil
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
dc.description.fil
Fil: Kues, Wilfried. Institut für Nutztiergenetik; Alemania
dc.journal.title
Reproduction Fertility and Development
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/http://dx.doi.org/10.1071/RDv27n1Ab356
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/http://www.publish.csiro.au/rd/RDv27n1Ab356


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