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Related Experiment Videos

An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences.

A Jalanko1, A Kallio, M Ruohonen-Lehto

  • 1Recombinant DNA Laboratory, University of Helsinki, Finland.

Biochimica Et Biophysica Acta
|February 28, 1988
PubMed
Summary
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A new mammalian cell expression vector utilizes human cytomegalovirus enhancer and Epstein-Barr virus elements for high-level gene expression. The vector DNA replicates extrachromosomally, showing a correlation between copy number and expression in human and monkey cells.

Area of Science:

  • Molecular Biology
  • Gene Expression
  • Mammalian Cell Culture

Background:

  • Development of efficient mammalian expression vectors is crucial for biotechnology and research.
  • Existing vectors often face limitations in replication and sustained gene expression.
  • Human cytomegalovirus (CMV) and Epstein-Barr virus (EBV) elements offer potential for enhanced vector function.

Purpose of the Study:

  • To construct and characterize a novel mammalian cell expression vector.
  • To evaluate the gene expression capacity and replication properties of the new vector.
  • To assess the correlation between vector copy number and transgene expression levels.

Main Methods:

  • Construction of an expression vector incorporating human cytomegalovirus immediate early gene enhancer, EBV EBNA-1 gene, ori-P sequences, and hygromycin B resistance gene.

Related Experiment Videos

  • Insertion of the chloramphenicol acetyltransferase (CAT) gene to test expression.
  • Transfection of the EBV-CAT construct into various mammalian cell lines.
  • Quantification of CAT activity and determination of vector DNA copy number per cell.

    • Main Results:

    • High levels of chloramphenicol acetyltransferase (CAT) activity were achieved in human and monkey cells.
    • The vector DNA replicated as an extrachromosomal element, with 1 to 20 copies per cell.
    • A positive correlation was observed between vector copy number and the expression level of the inserted gene in different cell clones.

    Conclusions:

    • The novel mammalian expression vector demonstrates efficient gene expression and extrachromosomal replication in human and monkey cells.
    • The vector's performance is linked to its copy number, suggesting a controllable system for gene expression.
    • This construct represents a valuable tool for applications requiring high-level, sustained transgene expression in mammalian systems.