title: Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal.
abstract: There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.
authors.affiliation: Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg. hartwig.huemer@uibk.ac.at
authors.forename: H P
authors.initials: HP
authors.lastname: Huemer
authors.forename: B
authors.initials: B
authors.lastname: Strobl
authors.forename: H
authors.initials: H
authors.lastname: Shida
authors.forename: C P
authors.initials: CP
authors.lastname: Czerny
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002645:Chickens; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007739:L Cells; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011995:Recombination, Genetic; D014162:Transfection; D014613:Vaccines, Attenuated; D014616:Vaccinia virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins
chemical_list: D008164:Luminescent Proteins; D014613:Vaccines, Attenuated; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C096213:glycoprotein 13, Equid herpesvirus; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases