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Amplification using CHO cell expression vectors.

Robert E Kingston1, Randal J Kaufman, C R Bebbington

  • 1Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Current Protocols in Molecular Biology
|February 12, 2008
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Summary
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Two gene amplification systems, dihydrofolate reductase (DHFR) and glutamine synthetase (GS), enable stable cell line generation. The GS system offers faster maximal expression compared to the lengthy DHFR method.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Stable transfection requires integrating plasmid DNA into host chromosomes.
  • High-level gene expression is achieved by selecting for amplification of linked selectable markers.
  • Two primary systems, DHFR and GS, facilitate gene amplification and expression.

Purpose of the Study:

  • To present and compare two systems for gene amplification and expression: DHFR and GS.
  • To detail the methodologies for generating stably transfected cell lines with high gene expression.

Main Methods:

  • Description of the dihydrofolate reductase (DHFR) selection system.
  • Description of the glutamine synthetase (GS) gene selection system.
  • Comparison of selection strategies for gene amplification and stable cell line generation.

Main Results:

  • The DHFR system allows very high levels of gene amplification and expression but requires a lengthy selection process (months).
  • The GS system typically achieves maximal expression levels in a single round of selection, offering a faster approach.
  • Both systems enable the generation of stably transfected cell lines with high-level gene expression via selectable marker amplification.

Conclusions:

  • The GS system provides a more rapid method for achieving maximal gene expression compared to the DHFR system.
  • Selection of amplification systems is crucial for efficient generation of stably transfected cell lines with desired gene expression levels.
  • Both DHFR and GS systems are valuable tools in molecular biology for producing high-expressing cell lines.