Orientation-dependent gene expression with Epstein-Barr virus-derived vectors.
Episomal vectors, described for efficient and regulated expression of heterologous proteins in mammalian cells, have the advantage that they persist in multiple copies in the cell without integrating into the chromosome. To efficiently express heterologous proteins we used such a vector based on elements of the Epstein-Barr virus (EBV), namely the sequences coding for Epstein-Barr nuclear antigen 1 and the viral origin of replication. Because constitutive expression is often deleterious to the cell, we combined the interferon-inducible Mx promoter with this EBV-derived vector. This resulted in an efficient and strictly regulated expression of the reporter gene chloramphenicol acetyltransferase (CAT) and of the neurotransmitter receptor h5-HT(1B), reaching levels of 16 ng CAT/mg cytoplasmic protein and 1300 fmol receptor/mg membrane protein, respectively. For both proteins, the expression levels were influenced by the orientation of the expression cassette. The higher expression in the favored orientation did not result from a higher copy number of these episomes. Northern analysis revealed a transcriptional read-through from the thymidine kinase promoter on the episomal vector, which interfered with the transcription of the heterologous gene in the less favored orientation.
Van Craenenbroeck
Kathleen
K
Department for Molecular Biology, Ghent University - VIB, K.L. Ledeganckstraat 35, 9000, Gent, Belgium. guy.haegeman@ugent.be
Vanhoenacker
Peter
P
Roman
Inge
I
Haegeman
Guy
G
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D002522:Chlorocebus aethiops; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D044302:Receptor, Serotonin, 5-HT1B / Q000096:biosynthesis / Q000235:genetics; D018741:Replication Origin; D014158:Transcription, Genetic; D014162:Transfection; D014709:Vero Cells; D014779:Virus Replication
D019309:Epstein-Barr Virus Nuclear Antigens; D016898:Interferon-alpha; D044302:Receptor, Serotonin, 5-HT1B; D015500:Chloramphenicol O-Acetyltransferase