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Yashas Rajendra1,2,3

  • 1Laboratory of Cellular Biotechnology (LBTC), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. rajendra_yashas@lilly.com.

Methods in Molecular Biology (Clifton, N.J.)
|September 23, 2018
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Summary
This summary is machine-generated.

A new method uses polyethyleneimine (PEI) for transient transfection of Chinese hamster ovary (CHO-DG44) cells, enabling scalable protein expression from 2 mL to 2 L. This high-cell-density approach optimizes DNA and PEI concentrations for efficient gene delivery.

Keywords:
CHO cellsOrbital shakingPolyethyleneimineRecombinant proteinTransfection

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Molecular Biology

Background:

  • Transient transfection is crucial for recombinant protein production in mammalian cells.
  • Optimizing transfection protocols for suspension-adapted cells like CHO-DG44 is essential for scalability.
  • Existing methods may lack efficiency or scalability for diverse applications.

Purpose of the Study:

  • To develop and describe a robust, scalable method for polyethyleneimine (PEI)-mediated transient transfection of suspension-adapted CHO-DG44 cells.
  • To optimize transfection parameters for efficient protein expression across a wide volume range (2 mL to 2 L).
  • To provide practical guidelines for implementing the method in research and bioproduction settings.

Main Methods:

  • Transfection of CHO-DG44 cells at high density (5 × 10^6 cells/mL) using direct addition of plasmid DNA (pDNA) and PEI.
  • Incubation at 31°C with orbital shaking for optimal gene expression.
  • Utilized specific concentrations: 0.3 mg/L coding pDNA, 2.7 mg/L filler DNA, and 15 mg/L PEI.
  • Production phase conducted at 31°C with 0.25% N,N-dimethylacetamide (DMA).

Main Results:

  • Successfully demonstrated PEI-mediated transient transfection for protein expression in suspension CHO-DG44 cells.
  • The method is validated for scales ranging from 2 mL to 2 L.
  • Detailed information on culture vessels, working volumes, and shaking speeds is provided for different scales.

Conclusions:

  • The described method offers a scalable and efficient approach for transient gene expression in CHO-DG44 cells.
  • This protocol facilitates high-level protein production applicable to various research and biomanufacturing scales.
  • The optimization of transfection conditions and inclusion of practical guidance enhance its utility.