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Improved bicistronic mammalian expression vectors using expression augmenting sequence element (EASE).

T L Aldrich1, J N Thomas, A E Morris

  • 1Immunex Corporation, 51 University Street, Seattle, Washington, 98101, U.S.A.

Cytotechnology
|November 13, 2008
PubMed
Summary
This summary is machine-generated.

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The Expression Augmenting Sequence Element (EASE) speeds up the development of stable cell pools for high-level recombinant protein production. This DNA element facilitates tandem vector integration, reducing development time by up to four weeks.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Cell Line Development

Background:

  • Chinese hamster ovary (CHO) cells are widely used for recombinant protein production.
  • Development of stable CHO cell pools for high-level protein expression can be time-consuming.
  • Expression Augmenting Sequence Element (EASE) was previously identified to enhance protein expression.

Purpose of the Study:

  • To map the active region of the EASE DNA element.
  • To develop and evaluate bicistronic expression vectors containing a smaller EASE fragment.
  • To assess the impact of EASE on the time required for stable cell pool development and protein expression.

Main Methods:

  • Colony forming assays were used to map the functional EASE region to 2.1 kb.
  • Bicistronic expression vectors were constructed using the smaller EASE or control lambda DNA.

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  • Stable CHO cell pools expressing FLT3 Ligand were developed in methotrexate-containing media.
  • Southern blot analysis was performed to investigate vector integration patterns.

    • Main Results:

    • The active EASE region was mapped to a 2.1 kb DNA fragment.
    • EASE-containing vectors reduced the time to develop stable cell pools by 1-4 weeks compared to control vectors.
    • Both EASE and control vectors yielded similar final protein expression levels.
    • Southern blot analysis indicated tandem integration of EASE-containing vectors, while control vectors showed interrupted integration.

    Conclusions:

    • The 2.1 kb EASE fragment facilitates tandem integration of bicistronic expression vectors in CHO cells.
    • EASE significantly reduces the time required for developing stable cell pools for recombinant protein production.
    • EASE is a valuable tool for accelerating biopharmaceutical development using CHO cell expression systems.