title: High-level expression of a cDNA for human granulocyte colony-stimulating factor in Chinese hamster ovary cells. Effect of 3'-noncoding sequences.
abstract: 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 beta 1-globin gene showed that use of the sequences from the rabbit beta 1-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 beta 1-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 micrograms rhG-CSF/1 x 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 micrograms/1 x 10(6) cells in 24 h.
authors.affiliation: Department of Biotechnology, Menarini Ricerche S.p.A. Pomezia, Roma, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Rotondaro
authors.forename: L
authors.initials: L
authors.lastname: Mazzanti
authors.forename: A
authors.initials: A
authors.lastname: Mele
authors.forename: G
authors.initials: G
authors.lastname: Rovera
mesh_terms: D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D018076:DNA, Complementary; D005784:Gene Amplification; D015870:Gene Expression; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D005914:Globins; D013762:Tetrahydrofolate Dehydrogenase