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A plasmid that replicates in both mouse and E. coli cells.

P J Kushner, B B Levinson, H M Goodman

    Journal of Molecular and Applied Genetics
    |January 1, 1982
    PubMed
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    Researchers created a novel shuttle vector using bovine papilloma virus (BPV) DNA that efficiently transforms mouse cells, even with bacterial DNA sequences. This vector replicates in both mammalian cells and E. coli, enabling gene transfer and study.

    Area of Science:

    • Molecular Biology
    • Virology
    • Genetic Engineering

    Background:

    • Cloned bovine papilloma virus (BPV) DNA can transform cells and replicate as an extrachromosomal element.
    • BPV DNA is a potential replicon for creating shuttle vectors capable of replicating in both mammalian cells and E. coli.
    • Previous attempts at creating shuttle vectors were hindered by bacterial plasmid sequences disrupting cellular transformation.

    Purpose of the Study:

    • To construct a functional shuttle plasmid vector that includes BPV DNA, a gene of interest, and bacterial plasmid sequences.
    • To investigate whether this shuttle vector could efficiently induce cellular transformation in mammalian cells.
    • To assess the replication and stability of the shuttle vector in both mammalian cells and E. coli.

    Main Methods:

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    • Construction of a shuttle plasmid (pGP) containing the transforming region of BPV, the rat growth hormone gene, and the bacterial plasmid pBR 327.
    • Introduction of the pGP plasmid into mouse cells to assess cellular transformation efficiency.
    • Replication studies of pGP in both mouse cells and E. coli.
    • Analysis of gene expression from the introduced rat growth hormone gene in mouse cells.
    • Co-transfer experiments with the herpes simplex virus thymidine kinase (tk) gene.

    Main Results:

    • The intact pGP molecule, despite containing bacterial sequences, efficiently induced cellular transformation in mouse cells.
    • A similar plasmid lacking the growth hormone gene did not transform mouse cells, indicating the gene's importance for transformation.
    • The pGP molecule replicated stably as an episome in both mouse cells (30-80 monomer episomes/cell) and E. coli.
    • The rat growth hormone gene was transcribed in mouse cells, producing a transcript longer than authentic mRNA and not regulated by glucocorticoids.
    • Co-transfer of pGP with the tk gene into mouse L-cells resulted in integration of the plasmid, with no free episomes observed.

    Conclusions:

    • A novel, efficient shuttle vector (pGP) has been successfully constructed using BPV DNA, which overcomes previous limitations associated with bacterial sequences.
    • This vector facilitates high-efficiency cellular transformation and stable replication in both mammalian and bacterial systems.
    • The pGP vector serves as a valuable tool for gene transfer, replication, and expression studies in diverse biological systems.