PEI-Mediated Transient Gene Expression in CHO Cells.

Area of Science:

• Biotechnology
• Cell Biology
• Molecular Biology

Background:

• Transient transfection is crucial for recombinant protein production in mammalian cells.
• Optimizing transfection efficiency and scalability remains a key challenge for industrial applications.
• Chinese Hamster Ovary (CHO) cells are widely used for biopharmaceutical manufacturing.

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 conditions for high-level protein expression across a wide range of volumes (2 mL to 2 L).
• To provide detailed protocols for successful gene delivery and protein production.

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 transfection and protein production.
• Utilized specific concentrations of coding pDNA, filler DNA, and PEI, with optional N,N-dimethylacetamide (DMA) addition.

Main Results:

• Successfully demonstrated a scalable transfection method applicable from small-scale (2 mL) to large-scale (2 L) cultures.
• Identified optimal reagent concentrations: 0.3 mg/L coding pDNA, 2.7 mg/L filler DNA, and 15 mg/L PEI.
• Showcased a modified protocol to eliminate DMA by adjusting coding DNA levels, offering flexibility in production.

Conclusions:

• The described PEI-mediated transfection method offers a reliable and scalable approach for protein expression in CHO-DG44 cells.
• The protocol is adaptable for various culture volumes, facilitating process development and manufacturing.
• This method provides a valuable tool for efficient recombinant protein production in biopharmaceutical research and industry.