High-Level Expression of a cDNA for Human Granulocyte Colony-Stimulating Factor in Chinese Hamster Ovary Cells Effect of 3'-Noncoding Sequences

摘要

We compared the production of recombinant human granulocyle 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 pi-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 trans-fected into a CHO cell line, yielded initial transfectant CHO cell lines secreting up to 21 μg rhG-CSF/1 * 10~6 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 * 10~6 cells in 24 h.