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A replication-competent promoter-trap retrovirus

J Coulombe1, Y Avis, D A Gray

  • 1Department of Biochemistry, University of Ottawa, Ontario, Canada.

Journal of Virology
|October 1, 1996
PubMed
Summary
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Researchers developed a novel retrovirus (PyT) to detect active gene promoters in mouse mammary tumors. This tool successfully identified a new gene, TRAP1, by integrating near its promoter, demonstrating its utility in vivo.

Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • Identifying active gene promoters is crucial for understanding gene regulation and disease development.
  • Retroviral vectors are widely used tools for gene delivery and insertional mutagenesis studies.
  • The polyomavirus middle T antigen is known to induce tumors in various animal models.

Purpose of the Study:

  • To construct and validate a novel promoter-trap retrovirus for detecting active promoters in vivo.
  • To investigate the integration patterns and oncogenic potential of the developed retrovirus in mouse mammary glands.
  • To identify novel genes activated by retroviral insertion in tumor development.

Main Methods:

  • Construction of a promoter-trap retrovirus (PyT) by inserting a promoterless polyomavirus middle T antigen gene into the U3 region of a Moloney murine leukemia virus.

Related Experiment Videos

  • Infection of mouse mammary glands in situ with the PyT retrovirus.
  • Analysis of tumor formation and PyT proviral integration sites in infected animals.
  • Cloning and characterization of the PyT provirus and flanking genomic sequences from tumor DNA.

    • Main Results:

    • PyT retrovirus induced mammary tumors in some infected mice.
    • PyT proviruses were found integrated within the tumors with the expected structure.
    • Integration occurred adjacent to the promoter of a novel mouse gene, TRAP1, which showed low-level expression in various tissues.
    • The PyT retrovirus demonstrated sensitivity in detecting active endogenous promoters in vivo.

    Conclusions:

    • The PyT retrovirus is an effective tool for identifying active gene promoters in a physiological context.
    • Retroviral insertional mutagenesis using PyT can lead to the discovery of novel, low-expressed genes involved in tumorigenesis.
    • This approach provides a sensitive method for promoter discovery and functional genomics in vivo.