A GFP-based screen for growth-arrested, recombinant protein-producing cells

Area of Science:

• Biotechnology
• Cell Biology
• Bioprocessing

Background:

• Mammalian cell culture, particularly Chinese hamster ovary (CHO) cells, is crucial for recombinant protein production.
• Stable expression of therapeutic proteins in CHO cells is challenging due to low transfection efficiency and selection difficulties.
• Serum deprivation typically arrests CHO cell growth, impacting protein production.

Purpose of the Study:

• To develop a rapid strategy for generating and selecting robust CHO cell subpopulations for recombinant protein production under serum-free conditions.
• To overcome limitations of traditional transformation and selection methods for stable protein expression.
• To identify CHO cell variants that maintain viability and therapeutic protein production during growth arrest.

Main Methods:

• Utilized bicistronic retroviral expression constructs encoding interferon gamma (IFN-gamma) and green fluorescent protein (GFP).
• Infected susceptible CHO cells with recombinant retroviruses, selecting for integrated expression modules via fluorescence-activated cell sorting (FACS).
• Applied serum deprivation as a selection pressure, followed by isolation and expansion of surviving GFP-expressing cells using FACS.

Main Results:

• Successfully generated and isolated a heterogeneous CHO cell population using bicistronic retroviral technology.
• Demonstrated that selected CHO cell subpopulations remained robust and continued stable expression of GFP and IFN-gamma during extended serum-free culture.
• Achieved isolation of cells for recombinant protein production under serum-free conditions within two weeks.

Conclusions:

• The developed strategy efficiently selects for CHO cell subpopulations capable of sustained recombinant protein production under serum-free conditions.
• Bicistronic retroviral expression coupled with FACS offers a rapid and effective method for isolating high-producing, robust cell lines.
• This approach significantly improves the process of generating stable mammalian cell lines for biopharmaceutical manufacturing.