Sodium Chloride → Tricalcium Phosphate

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반응 단계

The CHO cell expression vector, pCAGGS-DHFR/HGF was introduced into a DHFR-defective cell of Chinese hamster CHO cells by the method of Wigler et al. [Cell, 11, p233 (1977)]. About 30 μg of the pCAGGS-DHFR/HGF plasmid was dissolved in each 240 μL of 0.5 M calcium chloride, and 240 μL of a 2×HEPES buffer (pH 7.1) comprising 20 mM HEPES, 280 mM sodium chloride and 1.5 mM sodium phosphate was added with stirring. Stirring was continued at room temperature for 30 minutes to form a coprecipitate of the plasmid and calcium phosphate. Subsequently, 5×105 CHO cells were cultured at 37° C. for 24 hours under 5% CO2 using an α-MEM medium (Flow Laboratory) containing 10% bovine fetal serum (Gibco) and 1% glutamine. After medium exchange, the coprecipitate of the plasmid and calcium phosphate was added, and this was allowed to stand at room temperature for 20 minutes. Further, after incubation at 37° C. for 4 hours, the medium was removed, a 1×HEPES buffer with 15% glycerin added thereto was added, and this was allowed to stand at room temperature for 5 minutes. After cells were washed with a medium, the medium was exchanged, and this was cultured at 37° C. for 7 days to obtain a transformed cell. The resulting cell strain did not contain a ribonucleoside and a deoxyribonucleoside, and in order to obtain a stable HGF-highly producing strain using an α-MEM medium (Flow Laboratory) containing dialyzed 10% bovine fetal serum (Gibco) and 2% glutamine, passage culturing was repeated on the same medium by successively increasing a methotrexate concentration of 100 nM, 250 nM, 500 nM, 750 nM, 1 μM and 2 μM. Clone selection of the resulting HGF-producing recombinant cells was performed to obtain a stable HGF-producing strain.

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