title: Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability.
abstract: The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences, we deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFP, and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which were not different from those generated by the corresponding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors. However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene. These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene.
authors.affiliation: IST-Viral and Molecular Oncology Section, Via Gattamelata, 64 - 35128, Padua, Italy. stefano.indraccolo@unipd.it
authors.forename: Stefano
authors.initials: S
authors.lastname: Indraccolo
authors.forename: Valeria
authors.initials: V
authors.lastname: Roni
authors.forename: Rita
authors.initials: R
authors.lastname: Zamarchi
authors.forename: Francesca
authors.initials: F
authors.lastname: Roccaforte
authors.forename: Sonia
authors.initials: S
authors.lastname: Minuzzo
authors.forename: Laura
authors.initials: L
authors.lastname: Stievano
authors.forename: Walter
authors.initials: W
authors.lastname: Habeler
authors.forename: Novella
authors.initials: N
authors.lastname: Marcato
authors.forename: Veronica
authors.initials: V
authors.lastname: Tisato
authors.forename: Valeria
authors.initials: V
authors.lastname: Tosello
authors.forename: Luigi
authors.initials: L
authors.lastname: Chieco-Bianchi
authors.forename: Alberto
authors.initials: A
authors.lastname: Amadori
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011533:Proviruses / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D017384:Sequence Deletion; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins