High-level expression of a cDNA for human granulocyte colony-stimulating factor in Chinese hamster ovary cells
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We compared the production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by Chinese hamster ovary (CHO) cells in a transient expression system, using different analogous vectors carrying a human G-CSF-encoding cDNA under the transcriptional control of the murine cytomegalovirus (CMV) major immediate early promoter. Comparison of two transcription units carrying a human (h)G-CSF cDNA deleted of 3′-untranslated (UTR) sequences containing AT-rich elements (ARE) and using 3′-UTR sequences for processing of transcripts from the SV40 early region or from the rabbit β1-globin gene showed that use of the sequences from the rabbit β1-globin gene resulted in 7- to 12-fold higher levels of rhG-CSF production. Deletion of ARE of hG-CSF cDNA resulted in increased rhG-CSF synthesis when transcription units using 3′-UTR sequences from the rabbit β1-globin gene were compared. By contrast, deletion of ARE did not appear to affect rhG-CSF production when 3′-UTR sequences from the SV40 early region were used. The most efficient G-CSF transcription unit, fused to a dihydrofolate reductase (DHFR) marker gene and transfected into a CHO cell line, yielded initial transfectant CHO cell lines secreting up to 21 μg rhG-CSF/1 ×106 cells in 24 h. After two rounds of DHFR gene amplification, a cell line was isolated that contains approx 12 copies of the vector and produces rhG-CSF at a rate of 90 μg/1 × 106 cells in 24 h.
Index EntriesRecombinant DNA plasmid vectors transfection gene amplification cytokine AT-rich elements
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