Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production

Autores
Mufarrege, Eduardo Federico; Antuña, Sebastián; Marina Echeverrigaray; Kratje, Ricardo Bertoldo; Prieto, Claudio
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background: Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 50UTR (50 untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. Results: By combining the human cytomegalovirus (CMV) or the elongation factor 1a (EF-1a) promoters along with different 50UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 50UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1a promoter in three widely used cell lines. Conclusion: In the present work, LVs containing different promoters and 50UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.
Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Antuña, Sebastián. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina
Fil: Marina Echeverrigaray. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Materia
Cmv Enhancer/Promoter
Ef-1α Promoter
Lentiviral Vector
Protein Overexpression
Rhfviii Production
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:11336/31208
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/31208
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) productionMufarrege, Eduardo FedericoAntuña, SebastiánMarina EcheverrigarayKratje, Ricardo BertoldoPrieto, ClaudioCmv Enhancer/PromoterEf-1α PromoterLentiviral VectorProtein OverexpressionRhfviii Productionhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Background: Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 50UTR (50 untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. Results: By combining the human cytomegalovirus (CMV) or the elongation factor 1a (EF-1a) promoters along with different 50UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 50UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1a promoter in three widely used cell lines. Conclusion: In the present work, LVs containing different promoters and 50UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Antuña, Sebastián. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Marina Echeverrigaray. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaElsevier2013-11info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/31208Antuña, Sebastián; Mufarrege, Eduardo Federico; Prieto, Claudio; Kratje, Ricardo Bertoldo; Marina Echeverrigaray; Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production; Elsevier; Protein Expression and Purification; 95; 11-2013; 50-561046-5928CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2013.11.005info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046592813002428info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:02:33Zoai:ri.conicet.gov.ar:11336/31208instacron: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 10:02:33.508CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production
title Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production
spellingShingle Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production
Mufarrege, Eduardo Federico
Cmv Enhancer/Promoter
Ef-1α Promoter
Lentiviral Vector
Protein Overexpression
Rhfviii Production
title_short Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production
title_full Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production
title_fullStr Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production
title_full_unstemmed Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production
title_sort Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production
dc.creator.none.fl_str_mv Mufarrege, Eduardo Federico
Antuña, Sebastián
Marina Echeverrigaray
Kratje, Ricardo Bertoldo
Prieto, Claudio
author Mufarrege, Eduardo Federico
author_facet Mufarrege, Eduardo Federico
Antuña, Sebastián
Marina Echeverrigaray
Kratje, Ricardo Bertoldo
Prieto, Claudio
author_role author
author2 Antuña, Sebastián
Marina Echeverrigaray
Kratje, Ricardo Bertoldo
Prieto, Claudio
author2_role author
author
author
author
dc.subject.none.fl_str_mv Cmv Enhancer/Promoter
Ef-1α Promoter
Lentiviral Vector
Protein Overexpression
Rhfviii Production
topic Cmv Enhancer/Promoter
Ef-1α Promoter
Lentiviral Vector
Protein Overexpression
Rhfviii Production
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Background: Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 50UTR (50 untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. Results: By combining the human cytomegalovirus (CMV) or the elongation factor 1a (EF-1a) promoters along with different 50UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 50UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1a promoter in three widely used cell lines. Conclusion: In the present work, LVs containing different promoters and 50UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.
Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Antuña, Sebastián. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina
Fil: Marina Echeverrigaray. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
description Background: Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 50UTR (50 untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. Results: By combining the human cytomegalovirus (CMV) or the elongation factor 1a (EF-1a) promoters along with different 50UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 50UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1a promoter in three widely used cell lines. Conclusion: In the present work, LVs containing different promoters and 50UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.
publishDate 2013
dc.date.none.fl_str_mv 2013-11
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/11336/31208
Antuña, Sebastián; Mufarrege, Eduardo Federico; Prieto, Claudio; Kratje, Ricardo Bertoldo; Marina Echeverrigaray; Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production; Elsevier; Protein Expression and Purification; 95; 11-2013; 50-56
1046-5928
CONICET Digital
CONICET
url http://hdl.handle.net/11336/31208
identifier_str_mv Antuña, Sebastián; Mufarrege, Eduardo Federico; Prieto, Claudio; Kratje, Ricardo Bertoldo; Marina Echeverrigaray; Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production; Elsevier; Protein Expression and Purification; 95; 11-2013; 50-56
1046-5928
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2013.11.005
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046592813002428
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
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
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 13.070432

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