Measurable Parameters of Cells and Precipitate Predict Transfectability with Calcium Phosphate

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.