We have recently developed a method for efficient DNA transfection of mouse lymphoid cell lines, in which cells were first incubated with DNA-DEAE-dextran complex and then treated sequentially with hypertonic and hypotonic solutions (Takai and Ohmori, Biochim. Biophys. Acta 1990; 1048:105-109). We further attempted to improve this procedure, and found that the addition of dimethyl sulfoxide (DMSO) to the hypertonic solution resulted in a synergistic enhancement of the transfection frequencies. The cells that had been incubated with DNA and DEAE-dextran for 30 minutes were treated for 10 minutes with a hypertonic solution containing 0.4 M sucrose, 8% polyethylene glycol 4000, 84 mM NaCl, 28 mM Tris HCl, pH 7.3, and 10% DMSO. In mouse myeloma P3-NSl/1-Ag4-1 cells, for example, this improved DEAE-dextran method exhibited transfection frequencies of 10% and 1 x 10-4 for transient β-galactosidase gene expression and stable gpt transformation, respectively. These values were 30- and 4-fold higher than those obtained by the previous procedure, which did not use DMSO for the transient gene expression and the stable transformation, respectively. This procedure was applicable to a wide variety of cell lines of hematopoietic origin as well as to those with adherent nature, and should be useful for DNA transfection of hematopoietic cell lines that were refractory to other methods.
|Number of pages
|Methods in Molecular and Cellular Biology
|Published - 1990