title: Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells. A new strategy for recombinant human FVIII (rhFVIII) production.
abstract: 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 5'UTR (5' 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 1α (EF-1α) promoters along with different 5'UTRs 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 5'UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1α promoter in three widely used cell lines.
  
  
  CONCLUSIONS
  In the present work, LVs containing different promoters and 5'UTRs 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.
authors.affiliation: Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 242, S3000ZAA Santa Fe, Argentina. Electronic address: mufarrege@fbcb.unl.edu.ar.
authors.forename: Eduardo Federico
authors.initials: EF
authors.lastname: Mufarrege
authors.affiliation: Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Sebastián
authors.initials: S
authors.lastname: Antuña
authors.affiliation: Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Marina
authors.initials: M
authors.lastname: Etcheverrigaray
authors.affiliation: Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Kratje
authors.affiliation: Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Claudio
authors.initials: C
authors.lastname: Prieto
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003587:Cytomegalovirus / Q000235:genetics; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020648:Peptide Elongation Factor 1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C078147:F8 protein, human; D005169:Factor VIII
keywords: CMV enhancer/promoter; EF-1α promoter; Lentiviral vector; Protein overexpression; rhFVIII production