ABSTRACT
We have introduced the human beta-interferon gene with its promoter region into murine B-cell and fibroblast cell lines via a Moloney murine leukemia virus (M-MuLV) vector and have studied the inducible expression of the beta-interferon gene as a function of the various retroviral vector designs. By deleting the enhancer within the 3' viral long terminal repeat (LTR), inserting the human beta-interferon gene, and varying placement of the immunoglobulin heavy chain enhancer, we were able to construct vectors which yielded proviruses with various cell type-specific regulation. One of the vectors (pT154) led to a greater than 21-fold increase in beta-interferon protein synthesis after viral infection in the two B-cell lines analyzed, while no inducibility was seen in the fibroblast cells. The data show that inducible beta-interferon expression within a MuLV vector was highly dependent on the absence of the viral enhancer region in the long terminal repeat and the orientation of the beta-interferon gene within the proviral transcriptional unit; the insertion of the immunoglobulin enhancer elevated both constitutive and (or) inducible expression of beta-interferon in B-cells but inhibited constitutive expression of this gene in fibroblasts.
Subject(s)
B-Lymphocytes/metabolism , Genetic Vectors/genetics , Immunoglobulin Heavy Chains/genetics , Interferon Type I/genetics , Leukemia Virus, Murine/genetics , Animals , B-Lymphocytes/drug effects , Cell Line , Cloning, Molecular , Cycloheximide/pharmacology , Enhancer Elements, Genetic , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Interferon Type I/biosynthesis , Mice , Mice, Inbred Strains , Repetitive Sequences, Nucleic Acid , TransfectionABSTRACT
In mouse cells induced with virus infection or dsRNA, the relative levels of alpha-4 interferon mRNA were higher than the levels of alpha-1 and alpha-6 mRNAs; the ratio between relative levels of alpha-4 and alpha-1 or alpha-6 mRNA was, however, dependent on the cell type. Recombinant plasmids, in which the expression of chloramphenicol acetyltransferase (CAT) gene was directed by the promoter regions of alpha-1, alpha-4 or alpha-6 interferon genes were constructed and their inducible expression was studied either in transient assay or in permanently transfected mouse cells. The highest levels of CAT activity and CAT mRNA were observed with alpha-4 CAT plasmid, while the expression of alpha-1 CAT was consistently higher than that coded by alpha-6 CAT plasmid; the ratio between CAT activities coded by alpha-4 CAT and alpha-1 CAT was dependent on cell type. However, in heterologous Vero cells, the transfected alpha-1 and alpha-4 genes were expressed constitutively, and the levels of mRNAs were comparable. These results show that the difference in the relative levels of individual alpha-1 and alpha-4 mRNAs reflects the transcriptional inducibility of the respective promoter regions.
