High-level expression of a cDNA for human granulocyte colony-stimulating factor in Chinese hamster ovary cells
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
Unable to display preview. Download preview PDF.
- 1.Linch, D. C., Scarffe, H., Proctor, S., Chopra, R., Taylor, P. R. A., Morgenstern, G., Cunningham, D., Burnett, A. K., Cawley, J. C., Franklin, I. M., Bell, A. J., Lister, T. A., Marcus, R. E., Newland, A. C., Parker, A. C., and Yver, A. (1993) Randomized vehicle-controlled dose-finding study of glycosylated recombinant granulocyte colony-stimulating factor after bone marrow transplantation.Bone Marrow Transplant. 11, 307–311.PubMedGoogle Scholar
- 3.Oh-eda, M., Hasegawa, M., Hattori, K., Kuboniwa, H., Kojima, T., Orita, T., Tomonou, K., Yamazaki, T., and Ochi, N. (1990) O-linked sugar chain of human granulocyte-colony stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity.J. Biol. Chem. 265, 11,432–11,435.Google Scholar
- 10.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
- 11.Tweardy, D. J., Cannizzaro, L. A., Palumbo, A. P., Shane, S., Huebner, K., Vantuinen, P., Ledbetter, D. H., Finan, J. B., Nowell, P. C., and Rovera, G. (1987) Molecular cloning and characterization of a cDNA for human granulocyte colony-stimulating factor (G-CSF) from a glioblastoma multiforme cell line and localization of the G-CSF gene to chromosome band 17q21.Oncogene Res. 1, 209–220.PubMedGoogle Scholar
- 12.Dorsch-Häsler, K., Keil, G. M., Weber, F., Jasin, M., Schaffner, W., and Koszinowski, U. H. (1985) A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus.Proc. Natl. Acad. Sci. USA 82, 8325–8329.PubMedCrossRefGoogle Scholar
- 17.Gibco BRL (1989) Composition of the 1kb DNA ladder.Focus 11, 36.Google Scholar
- 18.Asselbergs, F. A. M., Will, H., Wingfield, P., and Hirschi, M. (1986) A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein.J. Mol. Biol. 189, 401–411.PubMedCrossRefGoogle Scholar
- 21.Yamazaki, T., Nagata, S., and Tsuchiya, M. (1987) Human granulocyte colony stimulating factor. EP 0 220 520 A1.Google Scholar