OPTIMIZATION OF EXPERIMENTAL-VARIABLES INFLUENCING REPORTER GENE-EXPRESSION IN HEPATOMA-CELLS FOLLOWING CALCIUM-PHOSPHATE TRANSFECTION

We describe a highly efficient calcium phosphate transfection protocol capable of achieving 100% transfection efficiency of reporter genes t ransiently expressed in the human hepatoma cell lines HuH7 and HepG2, This procedure, a modification of that described by Chen and Okayama, is reliable, reproducible, and eliminates the requirement for the incl usion of cotransfected internal control plasmids, While Chen and Okaya ma described the pH of the 2x BBS (N,N-bis[2-hydroxyethyl]-2-aminoetha nesulfonic acid-buffered saline) and DNA concentration as being critic al factors for optimal transfection efficiency, we show that a reduced and strictly monitored standing time of the DNA/CaCl2/2x BBS cocktail prior to addition to cultured cells is essential for a particular com bination of pH and DNA concentration. We also show that the inclusion of internal control plasmids can inhibit reporter gene activity in a p romoter- and dose-dependent manner. The method so described is also ap plicable for the transfection of other mammalian cell lines including COS and HeLa, and conceivably for the generation of stable transfectan ts at high frequency.