Expression from cell type-specific enhancer-modified retroviral vectors after transduction: Influence of marker gene stability

Stefano Indraccolo, Valeria Roni, Rita Zamarchi, Francesca Roccaforte, Sonia Minuzzo, Laura Stievano, Walter Habeler, Novella Marcato, Veronica Tisato, Valeria Tosello, Luigi Chieco-Bianchi, Alberto Amadori

Research output: Contribution to journalArticle

12 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)199-208
Number of pages10
JournalGene
Volume283
Issue number1-2
DOIs
Publication statusPublished - Jan 23 2002

Fingerprint

Genes
Reporter Genes
enhanced green fluorescent protein
Moloney murine leukemia virus
Human Activities
Half-Life
Endothelial Cells
Cell Line

Keywords

  • CD2
  • Endothelial cells
  • Enhanced green fluorescent protein
  • Enhancer
  • Lymphocytes
  • Tie2

ASJC Scopus subject areas

  • Genetics

Cite this

Expression from cell type-specific enhancer-modified retroviral vectors after transduction : Influence of marker gene stability. / Indraccolo, Stefano; Roni, Valeria; Zamarchi, Rita; Roccaforte, Francesca; Minuzzo, Sonia; Stievano, Laura; Habeler, Walter; Marcato, Novella; Tisato, Veronica; Tosello, Valeria; Chieco-Bianchi, Luigi; Amadori, Alberto.

In: Gene, Vol. 283, No. 1-2, 23.01.2002, p. 199-208.

Research output: Contribution to journalArticle

Indraccolo, Stefano ; Roni, Valeria ; Zamarchi, Rita ; Roccaforte, Francesca ; Minuzzo, Sonia ; Stievano, Laura ; Habeler, Walter ; Marcato, Novella ; Tisato, Veronica ; Tosello, Valeria ; Chieco-Bianchi, Luigi ; Amadori, Alberto. / Expression from cell type-specific enhancer-modified retroviral vectors after transduction : Influence of marker gene stability. In: Gene. 2002 ; Vol. 283, No. 1-2. pp. 199-208.
@article{e383b4acee89400d888b8b4140d4cfb7,
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.",
keywords = "CD2, Endothelial cells, Enhanced green fluorescent protein, Enhancer, Lymphocytes, Tie2",
author = "Stefano Indraccolo and Valeria Roni and Rita Zamarchi and Francesca Roccaforte and Sonia Minuzzo and Laura Stievano and Walter Habeler and Novella Marcato and Veronica Tisato and Valeria Tosello and Luigi Chieco-Bianchi and Alberto Amadori",
year = "2002",
month = "1",
day = "23",
doi = "10.1016/S0378-1119(01)00857-5",
language = "English",
volume = "283",
pages = "199--208",
journal = "Gene",
issn = "0378-1119",
publisher = "Elsevier B.V.",
number = "1-2",

}

TY - JOUR

T1 - Expression from cell type-specific enhancer-modified retroviral vectors after transduction

T2 - Influence of marker gene stability

AU - Indraccolo, Stefano

AU - Roni, Valeria

AU - Zamarchi, Rita

AU - Roccaforte, Francesca

AU - Minuzzo, Sonia

AU - Stievano, Laura

AU - Habeler, Walter

AU - Marcato, Novella

AU - Tisato, Veronica

AU - Tosello, Valeria

AU - Chieco-Bianchi, Luigi

AU - Amadori, Alberto

PY - 2002/1/23

Y1 - 2002/1/23

N2 - 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.

AB - 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.

KW - CD2

KW - Endothelial cells

KW - Enhanced green fluorescent protein

KW - Enhancer

KW - Lymphocytes

KW - Tie2

UR - http://www.scopus.com/inward/record.url?scp=0037160532&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037160532&partnerID=8YFLogxK

U2 - 10.1016/S0378-1119(01)00857-5

DO - 10.1016/S0378-1119(01)00857-5

M3 - Article

C2 - 11867226

AN - SCOPUS:0037160532

VL - 283

SP - 199

EP - 208

JO - Gene

JF - Gene

SN - 0378-1119

IS - 1-2

ER -