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A gene trap vector system for identifying transcriptionally responsive genes

Nature Biotechnology volume 19, pages 579–582 (2001)Cite this article

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 protein1–nitroreductase2 (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 cells3 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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Figure 1: Construction and validation of GFNR.
Figure 2: Selection of traps in HGF-regulated genes.
Figure 3: Responsiveness of trapped clones to HGF.

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Acknowledgements

We gratefully acknowledge Richard Palmiter (University of Washington–Seattle) for providing NTR cDNA. Thanks to Giuseppe Basso, Massimo Geuna, and the Fred Hutchinson Cancer Research Center (FHCRC) flow cytometry resource staff for their help with flow cytometry and cell sorting. We thank Weisheng Chen, Jon Cooper, Guy Hamilton, Jeff Hildebrand, and Masayuki Komada for critical comments on the manuscript. During his stay at FHCRC, E.M. was supported by a short-term fellowship from Fondazione Italiana per la Ricerca sul Cancro (FIRC). This research was supported by grants HD24875 and HD25326 from NIH to P.S., and by grants from the Armenise-Harvard Foundation for Advanced Scientific Research and Associazione Italiana per la Ricerca sul Cancro (AIRC) to P.M.C.

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Authors and Affiliations

  1. Institute for Cancer Research and Treatment, University of Torino School of Medicine, Candiolo, 10060, Italy

    Enzo Medico, Giovanna Gambarotta, Alessandra Gentile & Paolo M. Comoglio

  2. Division of Basic Sciences, Program in Developmental Biology, Fred Hutchinson Cancer Research Center, Seattle, 98109, WA

    Enzo Medico & Philippe Soriano

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  1. Enzo Medico
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  2. Giovanna Gambarotta
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  4. Paolo M. Comoglio
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  5. Philippe Soriano
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Correspondence to Enzo Medico.

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Medico, E., Gambarotta, G., Gentile, A. et al. A gene trap vector system for identifying transcriptionally responsive genes. Nat Biotechnol 19, 579–582 (2001). https://doi.org/10.1038/89343

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