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Orientation‐dependent gene expression with Epstein‐Barr virus‐derived vectors
Author(s) -
Van Craenenbroeck Kathleen,
Vanhoenacker Peter,
Roman Inge,
Haegeman Guy
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01311-5
Subject(s) - biology , chloramphenicol acetyltransferase , heterologous , microbiology and biotechnology , expression vector , t7 rna polymerase , reporter gene , heterologous expression , gene , plasmid , gene expression , herpes simplex virus , transcription (linguistics) , expression cassette , virus , vector (molecular biology) , virology , recombinant dna , genetics , bacteriophage , escherichia coli , linguistics , philosophy
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.