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 transiently 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 inclusion of cotransfected internal control plasmids. While Chen and Okayama described the pH of the 2x BBS (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid-buffered saline) and DNA concentration as being critical 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 combination of pH and DNA concentration. We also show that the inclusion of internal control plasmids can inhibit reporter gene activity in a promoter- and dose-dependent manner. The method so described is also applicable for the transfection of other mammalian cell lines including COS and HeLa, and conceivably for the generation of stable transfectants at high frequency.
O'Mahoney
J V
JV
Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia.
Adams
T E
TE
D002130:Calcium Phosphates; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D002130:Calcium Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D008156:Luciferases; D001616:beta-Galactosidase; C485829:calcium phosphate, dibasic, anhydrous