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Published on: September 28, 2016
Transient gene expression in CHO cells monitored with automated flow cytometry
Greg Sitton1, Ann Hansgate, Friedrich Srienc
1Department of Chemical Engineering and Materials Science, University of Minnesota, 151 Amundson Hall, 421 Washington Avenue S.E., Minneapolis, MN, 55455-0312, USA.
Cytotechnology
|November 13, 2008
Summary
Transient gene expression in cultured cells shows a stepwise decrease in protein levels per cell due to plasmid partitioning. Automated flow cytometry enables bioreactor monitoring and control for enhanced protein production.
Area of Science:
- Biotechnology
- Cell Biology
- Bioprocess Engineering
Background:
- Transient gene expression is vital for industrial protein production and gene therapy.
- Quantitative assessment of expression kinetics is crucial for optimizing cell culture conditions.
- Previous studies observed an exponential decrease in protein expression per cell post-transfection.
Purpose of the Study:
- To investigate the kinetics of transient gene expression at the single-cell level.
- To understand the mechanisms behind the decrease in protein expression after transfection.
- To explore the utility of automated flow cytometry for bioreactor monitoring.
Main Methods:
- Utilized automated flow cytometry to monitor single-cell expression of enhanced Green Fluorescent Protein (eGfp).
- Analyzed cell physiology and protein content over time following electroporation.
- Quantified plasmid and eGfp partitioning in dividing cells.
Main Results:
- Demonstrated that eGfp expression decrease occurs in discrete steps, not exponentially.
- Attributed the stepwise decrease to the absence of plasmid replication and symmetric partitioning.
- Observed distinct cell physiological shifts and two unique stationary phases during culture recovery.
- Showcased constant increase in total eGfp within the bioreactor.
Conclusions:
- Stepwise decrease in single-cell eGfp expression is linked to plasmid partitioning dynamics.
- Automated flow cytometry provides real-time insights into cell physiology and culture status.
- Findings support the US FDA's Process Analytical Technology initiative for bioreactor control.
