Enhancing the efficiency of DNA-mediated gene transfer in mammalian cells

We have investigated several of the experimental factors that affect calcium phosphate-DNA-mediated gene transfer of thymidine kinase (tk) into mouse LM tk- Cl 1D cells using unfractionated DNA from both Chinese hamster ovary cells and L6 rat myoblasts. Increases in the length of exposure to DNA (24 h) and the expression time (48 h) before selection result in a 20-fold enhancement in the efficiency of transformation. These modifications yield frequencies up to 35 HATR colonies/20 microgram tk"NA/10(6) recipient cells. Exposure to dimethyl sulfoxide enhances transformation efficiencies slightly for short DNA exposure times, but has no effect when optimal DNA exposure times are used. Several other variations in our standard transformation protocol were also examined: these include the concentration and size of the DNA and exposure to low concentrations of the nonionic detergent, Tween-80. We have also isolated and characterized a subclone of Cl 1D that is a high-efficiency recipient for the tk+ marker. Segregation analysis reveals that the majority of the TK+ transformants derived from this subclone are stable, in contrast to those derived from the DL 1D parent. The combination of improved methodology and the high-efficiency recipient subclone permits DNA-mediated transformation for tk at frequencies on the order of 10(-4) transformants per recipient cell.