A gene trap vector system for identifying transcriptionally responsive genes

We present a method for fast and efficient trapping of genes whose transcri ption is regulated by exogenous stimuli. We constructed a promoterless retr oviral vector transducing a green fluorescent protein(1)-nitroreductase(2) (GFNR) fusion protein downstream from a splice acceptor site. Flow cytometr ic 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 selectio n against constitutive GFNR expression. Using hepatocyte growth factor (HGF ) stimulation of liver cells(3) combined with either positive or negative s election. we recovered cell populations carrying traps in induced or suppre ssed genes, respectively. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA leve l. Positive and negative selection can be calibrated to recover traps in ge nes showing different levels of basal expression or transcriptional regulat ion. The flexibility and efficiency of the GFNR-based trap screening proced ure make it suitable for wide surveys of transcriptionally regulated genes.