Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability.
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.
Indraccolo
Stefano
S
IST-Viral and Molecular Oncology Section, Via Gattamelata, 64 - 35128, Padua, Italy. stefano.indraccolo@unipd.it
Roni
Valeria
V
Zamarchi
Rita
R
Roccaforte
Francesca
F
Minuzzo
Sonia
S
Stievano
Laura
L
Habeler
Walter
W
Marcato
Novella
N
Tisato
Veronica
V
Tosello
Valeria
V
Chieco-Bianchi
Luigi
L
Amadori
Alberto
A
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
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins