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Related Experiment Videos

New host cell system for regulated simian virus 40 DNA replication.

R D Gerard, Y Gluzman

    Molecular and Cellular Biology
    |November 1, 1985
    PubMed
    Summary

    New monkey cell lines (CMT and BMT) can be induced to produce simian virus 40 (SV40) T antigen. These cells efficiently maintain and amplify cloned genes in an episomal state, useful for genetic studies.

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    Area of Science:

    • Molecular Biology
    • Virology
    • Cell Biology

    Background:

    • Development of inducible cell lines for studying viral gene expression.
    • Characterization of simian virus 40 (SV40) T antigen function.
    • Metallothionein promoter system for controlled gene expression.

    Purpose of the Study:

    • To isolate and characterize inducible monkey cell lines (CMT and BMT) expressing SV40 T antigen.
    • To investigate the correlation between T antigen levels and SV40 DNA replication.
    • To assess the utility of these cell lines for maintaining and amplifying cloned eukaryotic genes.

    Main Methods:

    • Isolation and characterization of transformed monkey cell lines (CMT and BMT).
    • Inducible expression of SV40 T antigen using the metallothionein promoter.
    • Immunoprecipitation and radioimmunoassay for quantifying T antigen synthesis and accumulation.
    • Transformation with a vector containing SV40 origin of replication and neomycin resistance marker.
    • Analysis of episomal maintenance and gene amplification.

    Main Results:

    • Heavy-metal inducers increased SV40 T antigen synthesis 5- to 12-fold and total accumulation 3- to 4-fold.
    • A direct correlation was observed between T antigen levels and SV40 DNA replication.
    • Inducible BMT cells efficiently maintained vector sequences in an episomal form, even without selection.
    • Heavy-metal-dependent amplification of the template increased expression of proteins encoded by SV40 late promoter-driven vectors.

    Conclusions:

    • Inducible monkey cell lines (CMT and BMT) provide a system for controlled SV40 T antigen expression.
    • These cell lines can efficiently maintain cloned eukaryotic genes in an amplifiable episomal state.
    • The system is valuable for studying gene regulation, DNA replication, and gene amplification in mammalian cells.

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