pMmEG(TA)-rhBMP-4

Protocol tips

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.

Publication protocol

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. In order to evaluate the furin expression on the processing of the expressed rhBMP-4, suspension adapted CHO DG44 cells with recombinant furin expression were prepared as described previously [20] and were used as the host for rhBMP-4 expression. The two host CHO cells with and without recombinant furin expression were designated as CHO(+f) and CHO(-f), respectively. 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.

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