Optimizing conditions for calcium phosphate mediated transient transfection

Abstract

Background: Calcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombi- nant DNA into culture cells. However, multiple factors affecting the transfect efficiency are com- monly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research. Results: In this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate–DNA co-precipitation and on-cell treat- ments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition opti- mized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step