Inducible expression of amplified human beta interferon genes in CHO cells.

Abstract

Plasmid DNA containing the human beta-interferon (IFN-,B) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-4 after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-P gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 1010 U/liter medium in culture supernatants. Constitutive production of IFN-P at rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-1 had acquired resistance to cytotoxic antiviral effects of IFN-P. Two forms of human IFN-,B were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-P production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-,B production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-,-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-P.Interferons are secreted polypeptides that protect cells from virus infection. Two classes of human interferon (IFN) have been described: type I IFNs (IFN-ot, IFN-P) are induced by viral infections and are acid stable and type II IFN (IFN--y) is induced after stimulation of T-lymphocytes and is acid sensitive. IFNs of both types have been purified to homogeneity, and their biochemical and biological properties have been studied extensively (19,26). Genes coding for these polypeptides have been cloned, and their nucleotide sequences have been determined (5,8,9,12,15,17,27 Fig. 1.Next, the 838-bp TaqI fragment of the IFN-P gene, which on May 12, 2018 by guest

Inhibition of protein synthesis stimulates the transcription of human beta-interferon genes in Chinese hamster ovary cells.

“…This increase, however, was observed after 8-12 hr of treatment (i.e., at times when IFN mRNA concentrations had already returned to basal levels); thus, the significance of this delayed increase in transcription remains unclear. There is additional evidence, however, that the effect of poly(rI)poly(rC) on IFN production requires the IFN promoter region (12,27,28). When a simian virus 40 promoter is fused to the IFN-,8 coding region, the hybrid gene is not inducible by either cycloheximide or poly(rI)-poly(rC) (12).…”

“…There is additional evidence, however, that the effect of poly(rI)poly(rC) on IFN production requires the IFN promoter region (12,27,28). When a simian virus 40 promoter is fused to the IFN-,8 coding region, the hybrid gene is not inducible by either cycloheximide or poly(rI)-poly(rC) (12). Similarly, deletions that remove sequences upstream of the IFN-,B gene's start of transcription abrogate its inducibility by poly(rI)-poly(rC) (27,28).…”

“…We have recently reported that the human IFN-pB gene can be expressed in transfected Chinese hamster ovary (CHO) cells (12). Furthermore, the transfected gene retains inducibility in response to virus infection and can be superinduceq by poly(rI)-poly(rC) and by cycloheximide.…”

“…In contrast, when nuclear transcription products are hybrid- 10 ,g/ml, emetine at 10 ug/ml, puromycin at 50 ,sg/ml, and poly(rI) poly(rC) at 20 ,ug/ml. RNAs were analyzed by the dot blot procedure'using 2-fold serial dilutions of each sample as described (12). To assess whether differences in hybridization could be ascribed to differences in length of the [32P]RNAs, the nuclear transcription products were analyzed by polyacrylamide gel electrophoresis.…”

“…RNA was prepared from cells lysed in guanidine thiocyanate as described by Chirgwin et al (14) and analyzed on nitrocellulose dot blots as described (12). The probe used was the 1.…”