Efficient constitutive production of human fibroblast interferon by hamster cells transformed with the IFN-beta 1 gene fused to an SV40 early promoter.
The coding sequence of the human interferon (IFN)-beta 1 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-beta 1 into the medium. Induction did not stimulate expression of the fused SV40-IFN-beta 1 gene. The role of the SV40 promoter was verified by transforming cells with the unmodified human IFN-beta 1 gene, or by the IFN-beta 1 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-beta 1 DNA were obtained. Such clones constitutively produce up to 350,000 units IFN/ml per 10(6) 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-beta 1 is constant at a rate of 300,000 molecules per cell/hr. Batches of up to 16 mg of IFN-beta 1 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-beta 1 glycoprotein.
D000818:Animals; D003001:Cloning, Molecular; D006224:Cricetinae; D004274:DNA, Recombinant; D005784:Gene Amplification; D005786:Gene Expression Regulation; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis / Q000235:genetics*; D009876:Operon; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D007370:Interferon Type I