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Vaccinia virus: a selectable eukaryotic cloning and expression vector.

M Mackett, G L Smith, B Moss

    Proceedings of the National Academy of Sciences of the United States of America
    |December 1, 1982
    PubMed
    Summary
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    This study shows vaccinia virus can be used as a cloning and expression vector by inserting foreign DNA. Successful gene expression was confirmed using herpesvirus thymidine kinase.

    Area of Science:

    • Molecular Biology
    • Virology
    • Genetic Engineering

    Background:

    • Vaccinia virus is a large DNA virus with a well-characterized genome.
    • Developing efficient methods for genetic manipulation of vaccinia virus is crucial for its use as a vector.
    • Understanding vaccinia virus gene regulation is important for optimizing expression systems.

    Purpose of the Study:

    • To demonstrate the utility of vaccinia virus as a selectable cloning and expression vector.
    • To confirm the genomic map location of the vaccinia virus thymidine kinase (TK) gene.
    • To investigate vaccinia virus transcriptional regulatory sequences.

    Main Methods:

    • Homologous recombination was used to insert foreign DNA into nonessential regions of the vaccinia virus genome.

    Related Experiment Videos

  • Thymidine kinase-negative (TK-) recombinants were selected by disrupting the endogenous TK gene.
  • Thymidine kinase-positive (TK+) recombinants were generated by inserting the herpesvirus TK gene into TK- mutants.

    • Main Results:

    • Successful insertion and expression of foreign DNA into the vaccinia virus genome were achieved.
    • The map location of the vaccinia virus TK gene was confirmed.
    • Expression of the herpesvirus TK gene required specific upstream regulatory sequences from vaccinia virus.
    • Herpesvirus TK activity was confirmed by phosphorylation of 125I-labeled deoxycytidine.

    Conclusions:

    • Vaccinia virus serves as a versatile and selectable cloning and expression vector.
    • The study provides insights into vaccinia virus transcriptional regulatory elements.
    • This work facilitates further genetic engineering applications using vaccinia virus.