Efficient Constitutive Production of Human Fibroblast Interferon by Hamster Cells Transformed with the IFN-β1Gene Fused to An SV40 Early Promoter
- 1 August 1984
- journal article
- research article
- Published by Mary Ann Liebert Inc in DNA
- Vol. 3 (4), 297-308
- https://doi.org/10.1089/dna.1.1984.3.297
Abstract
The coding sequence of the human interferon (IFN)-β1 gene, fused 60 bp downstream from the RNA start site of the SV40 early gene, was transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells together with a selectable DHFR gene. Most transformants continuously secreted IFN-β1 into the medium. Induction did not stimulate expression of the fused SV40-IFN-β1 gene. The role of the SV40 promoter was verified by transforming cells with the unmodified human IFN-β1 gene, or by the IFN-β1 coding region fused to another poly(rI):(rC)-inducible gene. In these cases, the transformants showed strictly inducible (not constitutive) IFN secretion. By selection for methotrexate resistance, CHO clones with a 10–20-fold amplification of the SV40-IFN-β1 DNA were obtained. Such clones constitutively produce up to 350,000 units IFN/ml per 106 cells/24 hr, i.e., over 10 times more than fully induced human fibroblasts. In continuous culture with daily changes of medium, accumulation of IFN-β1 is constant at a rate of 300,000 molecules per cell/hr. Batches of up to 16 mg of IFN-β1 produced by the transformed CHO cells were purified to homogeneity by affinity chromatography on monoclonal antibodies. This IFN appears identical in size, activity, and immunospecificity to the native human IFN-β1 glycoprotein.Keywords
This publication has 49 references indexed in Scilit:
- Deletion mapping of the inducible promoter of human IFN-β geneNature, 1983
- Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA geneJournal of Molecular Biology, 1982
- Inducibility of human β-interferon gene in mouse L-cell clonesNature, 1982
- β+ Thalassemia: Aberrant splicing results from a single point mutation in an intronCell, 1981
- T antigen repression of SV40 early transcription from two promotersCell, 1981
- Synthesis of Human Interferon β1 in Escherichia coliInfected by a Lambda Phage Recombinant containing a Human Genomic FragmentEuropean Journal of Biochemistry, 1981
- A new dominant hybrid selective marker for higher eukaryotic cellsJournal of Molecular Biology, 1981
- Expression of human fibroblast interferon gene in Escherichia coliNature, 1980
- Identification of the Translation Products of Human Fibroblast Interferon mRNA in Reticulocyte LysatesEuropean Journal of Biochemistry, 1979
- Detection of specific sequences among DNA fragments separated by gel electrophoresisJournal of Molecular Biology, 1975