Abstract
Methods for the transfection of DNA into mammalian cells for transient gene expression were initially developed about 30 years ago. The main purpose was and still remains the molecular analysis of the cell’s response to the exogenous protein. The use of transient gene expression for industrial purposes began in 1991 when Bennett and coworkers identified by alanine-scanning mutagenesis several mutants of human tissue-plasminogen activator (rtPA) that were eventually shown to be of higher medical potency than the wild-type protein. One of the molecules, now an approved thrombolytic “TNKase™”, had a dramatically extended half-life in patients, a high fibrin-specificity, and was insensitive to the inhibitor of plasminogen-activator PAI. The identification of improved mutants was based on more than 40,000 transient gene expression experiments (Wurm et al. unpublished) executed during the course of one year using calcium-phosphate transfections in human embryonic kidney (HEK) 293 cells (Gorman et al 1990).
Only very recently has the usefulness of transient gene expression for the production of milligram or even gram quantities of protein been recognized. A number of insights and technological advances were necessary to permit transient gene expression in mammalian cells to be executed in operational scales up to 100 Liters. Only two systems have so far been developed for this scale, calcium-phosphate based transient transfection of HEK-293 cells (Girard et al. 2002) and a Genentech-proprietary lipid based system using genetically modified Chinese hamster ovary (CHO) cells. This paper will summarize in a cursory way the most important developmental steps and the underlying arguments that allowed transient gene expression to become a second way for industrial protein production in mammalian cells. By comparison, virus based transient expression systems (Baculovirus, Alphavirus) have been restricted up to now to operational scales below 10 Liters and may not advance much further in terms of scale and high-throughput activity because of a number of inherent complexities, the most restrictive being a limit on the size/quantity of DNA that can be delivered. Other technological and biological problems also limit scale-up.
Keywords
- Transfection Efficiency
- Chinese Hamster Ovary Cell
- Transient Transfection
- Sodium Butyrate
- Transient Gene Expression
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Batard, P., M. Jordan, P. Chatellard, F. Wurm (2001): Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection. Gene 270, 61–68
Bennett W.F., N. Paoni, D. Botstein, A.J.S. Jones, B. Keyt, L. Presta, F.M. Wurm and M. Zoller (1991): Functional Properties of a Collection of Charged-to-Alanine Substitution Variants of Tissue-Type Plasminogen Activator. J. Biol. Chemistry 266, 8, pp. 5191–5201.
Girard, P. M. Derouazi, G. Baumgartner, M. Bourgeois, M. Jordan, B. Jacko, F.M. Wurm (2002): 100 Liter-transient transfection. Cytotechnology 38, 1–2: 57–62.
Gorman, C.M., D.R. Gies, G. McCray (1990): Transient production of proteins using an adenovirus transformed cell line. DNA Protein Engineering Techniques 2, 1–28.
Grosjean, F., Batard, P., M. Jordan, F. M. Wurm (2002): S-phase synchronized CHO cells show elevated transfection efficiency and expression using CaPi. Cytotechnology 38,1–2: 57–62. Hunt, L., P. Batard, M. Jordan, F.M. Wurm (2002) Fluorescent proteins in animal cells for process development: Optimization of sodium butyrate treatment as an example. Biotechnology and Bioengineering, Vol. 77, No. 5, p 528–537
Jordan, M., A. Schallhorn and F.M. Wurm (1996): Transfecting mammalian cells: optimisation of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Research 24,4, 596–601. Jordan, M., C. Kohne and F.M. Wurm (1998): Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Cytotechnology, 26, pp. 39–47.
Pick, H. M., P. Meissner, A.K. Preuss, P. Tromba, H. Vogel and F.M. Wurm (2002): Balancing GFP reporter plasmid quantity in large scale transient transfections for recombinant anti-human Rhesus-D IgGl synthesis. Biotechnology and Bioengineering 79,6, 595–601
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Springer Science+Business Media Dordrecht
About this paper
Cite this paper
Wurm, F.M. et al. (2003). Transient Gene Expression: A Novel Mammalian Cell-Based Technology for Recombinant Protein Production. In: Yagasaki, K., Miura, Y., Hatori, M., Nomura, Y. (eds) Animal Cell Technology: Basic & Applied Aspects. Animal Cell Technology: Basic & Applied Aspects, vol 13. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-0726-8_15
Download citation
DOI: https://doi.org/10.1007/978-94-017-0726-8_15
Publisher Name: Springer, Dordrecht
Print ISBN: 978-90-481-6557-5
Online ISBN: 978-94-017-0726-8
eBook Packages: Springer Book Archive