We present a method for fast and efficient trapping of genes whose transcription is regulated by exogenous stimuli. We constructed a promoterless retroviral vector transducing a green fluorescent protein-nitroreductase (GFNR) fusion protein downstream from a splice acceptor site. Flow cytometric analysis of the infected population allows identification and sorting of cells in which the trap is integrated downstream from an active promoter. Conversely, the nitroreductase (NTR) moiety allows pharmacological selection against constitutive GFNR expression. Using hepatocyte growth factor (HGF) stimulation of liver cells combined with either positive or negative selection, we recovered cell populations carrying traps in induced or suppressed genes, respectively. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA level. Positive and negative selection can be calibrated to recover traps in genes showing different levels of basal expression or transcriptional regulation. The flexibility and efficiency of the GFNR-based trap screening procedure make it suitable for wide surveys of transcriptionally regulated genes.

A gene trap vector system for identifying transcriptionally responsive genes

MEDICO, Enzo;GAMBAROTTA, Giovanna;COMOGLIO, Paolo;
2001

Abstract

We present a method for fast and efficient trapping of genes whose transcription is regulated by exogenous stimuli. We constructed a promoterless retroviral vector transducing a green fluorescent protein-nitroreductase (GFNR) fusion protein downstream from a splice acceptor site. Flow cytometric analysis of the infected population allows identification and sorting of cells in which the trap is integrated downstream from an active promoter. Conversely, the nitroreductase (NTR) moiety allows pharmacological selection against constitutive GFNR expression. Using hepatocyte growth factor (HGF) stimulation of liver cells combined with either positive or negative selection, we recovered cell populations carrying traps in induced or suppressed genes, respectively. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA level. Positive and negative selection can be calibrated to recover traps in genes showing different levels of basal expression or transcriptional regulation. The flexibility and efficiency of the GFNR-based trap screening procedure make it suitable for wide surveys of transcriptionally regulated genes.
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E. MEDICO; G. GAMBAROTTA; A. GENTILE; P. COMOGLIO; P. SORIANO
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/43351
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