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Related Experiment Videos

High spontaneous mutation frequency of BPV shuttle vector.

C R Ashman, R L Davidson

    Somatic Cell and Molecular Genetics
    |September 1, 1985
    PubMed
    Summary

    Mammalian cell passage induced mutations in bovine papillomavirus (BPV) shuttle vectors. Most mutated plasmids showed significant DNA rearrangements, suggesting genetic instability after mammalian cell exposure.

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    Area of Science:

    • Molecular Biology
    • Virology
    • Genetics

    Background:

    • Mammalian cells can be used to propagate and study viral genomes.
    • Shuttle vectors allow for replication and manipulation in both prokaryotic and eukaryotic systems.
    • Understanding genetic alterations in plasmids after passage is crucial for molecular biology applications.

    Purpose of the Study:

    • To investigate the genetic stability of a bovine papillomavirus (BPV) shuttle vector after propagation in mouse cells.
    • To quantify mutations in the Escherichia coli gpt gene within the shuttle vector.
    • To characterize DNA structural changes in mutated plasmids.

    Main Methods:

    • Construction of a recombinant shuttle vector containing BPV, pBR322, and E. coli gpt gene.
    • Transformation of mouse C127 cells with the shuttle vector.
    • Plasmid extraction from transformed mammalian cells.
    • Transformation of E. coli HB101 (Gpt-) with extracted plasmids.
    • Determination of mutant frequencies and restriction digest analysis.

    Main Results:

    • Plasmids passaged through mammalian cells exhibited approximate mutant frequencies of 3-16 X 10(-3).
    • Restriction digest analysis revealed gross DNA rearrangements in the majority of mutated plasmid molecules.
    • The gpt gene was used as a reporter for mutations.

    Conclusions:

    • Passage through mammalian cells induces significant genetic instability in shuttle vectors.
    • Mammalian cell environments can lead to substantial DNA structural alterations in plasmids.
    • These findings have implications for using shuttle vectors in gene expression and genetic studies.

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