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| Upstream tips |
Protocol tips |
Downstream tips |
| Suspension adapted CHO DG44 (dhfr- /dhfr- ) cells, adapted to the chemically defined medium, EX CELL CD CHO (Sigma-Aldrich, USA) supplemented with 4 mM L-glutamine (Sigma-Aldrich), were used as the host for rhBMP-4 expression. |
The CHO(+f) and CHO(-f) cells were co-transfected with 2 μg of DNA that consisted of a 100:1 molar ratio of the expression plasmid, pMmEG(TA)-rhBMP-4, and the selection marker plasmid, pDCH1P [21], by electroporation using the Neon transfection system (Life Technologies, USA) in accordance with the manufacturer’s instructions. |
After transfection, the pools of transfectants were grown in the selection medium, EX-CELL CD CHO (without hypoxanthine and thymidine) (SigmaAldrich) supplemented with 4mM L glutamine. The single-cellderived clones were isolated from pools of transfectants by limiting dilutions. |
| Upstream tips |
| Suspension adapted CHO DG44 (dhfr- /dhfr- ) cells, adapted to the chemically defined medium, EX CELL CD CHO (Sigma-Aldrich, USA) supplemented with 4 mM L-glutamine (Sigma-Aldrich), were used as the host for rhBMP-4 expression. |
| Protocol tips |
| The CHO(+f) and CHO(-f) cells were co-transfected with 2 μg of DNA that consisted of a 100:1 molar ratio of the expression plasmid, pMmEG(TA)-rhBMP-4, and the selection marker plasmid, pDCH1P [21], by electroporation using the Neon transfection system (Life Technologies, USA) in accordance with the manufacturer’s instructions. |
| Downstream tips |
| After transfection, the pools of transfectants were grown in the selection medium, EX-CELL CD CHO (without hypoxanthine and thymidine) (SigmaAldrich) supplemented with 4mM L glutamine. The single-cellderived clones were isolated from pools of transfectants by limiting dilutions. |
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