Abstract
Transient expression of proteins in mammalian cells at 1–100 L scale has the potential to become a powerful technique for the rapid production of research proteins. We have shown that the calcium phosphate technique is compatible with routine handling in bioreactors and that the yield of proteins is comparable to that seen in standard transient transfections1. However, although the reproducibility of the method is excellent within triplicates, there can be dramatic variations in transfection efficiencies with different batches of cells, solutions, plasmid preparations etc. In addition, expression levels of novel proteins may differ from those seen with proteins like tPA or hGH. There is a need therefore, to monitor the transfection procedure itself and to predict protein expression through defined and quantitative assays. We suggest two tests: the first assesses, through OD-measurements of the transfection cocktail, the quality of the DNA-carrier complexes. The second employs a commercially available vector encoding a strongly fluorescent green fluorescent protein (GFP). Transfection with this vector provides, within a day, a quantitative signal. Together, these assays allow a fast assessment of transfections with calcium phosphate for optimization purposes. Moreover, co-transfecting GFP with the vectors of interest allows, to a certain degree, prediction of transfectability and expression of the desired protein.
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References
Jordan, M., Koehne, C. and Wurm, F.M. (1997) Calcium phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Cytotcchnology in press
Jordan, M. and Wurm, F.M. (1995) High level transient expression in mammalian cells: identification and optimization of physico-chemical parameters of the calcium phosphate transfection method, in E.C. Beuvery et al.(eds.) Animal Cell Technology:Developments towards the 21st Century, Kluver Academic Publisher, Dordrecht. 49–55
Jordan, M., Schallhorn, A. and Wurm, F.M. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24. 596–601
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© 1998 Kluwer Academic Publishers
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Jordan, M., Wurm, F. (1998). Measurable Parameters of Cells and Precipitate Predict Transfectability with Calcium Phosphate. In: Merten, OW., Perrin, P., Griffiths, B. (eds) New Developments and New Applications in Animal Cell Technology. Springer, Dordrecht. https://doi.org/10.1007/0-306-46860-3_22
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DOI: https://doi.org/10.1007/0-306-46860-3_22
Publisher Name: Springer, Dordrecht
Print ISBN: 978-0-7923-5016-3
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