title: Optimization of baculovirus transduction on FreeStyle293 cells for the generation of influenza B/Lee/40.
abstract: Recombinant baculovirus expression vectors derived from the Autographa californica nuclear polyhedrosis virus can serve as efficient gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types and are able to produce multisubunit particles such as viruses or virus like particles. In this study, we constructed eight recombinant baculoviruses each containing one of the influenza B/Lee/40 virus genes in a bidirectional expression cassette for simultaneous mRNA and viral RNA transcription. Baculoviruses were transduced into FreeStyle293 in combination with the specific histone deacetylase inhibitor trichostatin A (TSA). Cotransduction conditions were optimized with a set of five baculoviruses (influenza B/Lee/40 PB1, PB2, PA, and NP and the control construct NCR-NS-minus-sense orientated encoding green fluorescent protein [rGFP]), which led to GFP expression in each host cell transduced with all five constructs. Based on the optimization with five constructs, transduction with eight baculoviruses was performed at MOI 50 and 100 with high yield stocks and 1 microM TSA and led to successful rescue of infectious influenza B/Lee/40 viruses.
authors.affiliation: Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria. sabine.nakowitsch@boku.ac.at
authors.forename: Sabine
authors.initials: S
authors.lastname: Nakowitsch
authors.forename: C
authors.initials: C
authors.lastname: Kittel
authors.forename: W
authors.initials: W
authors.lastname: Ernst
authors.forename: A
authors.initials: A
authors.lastname: Egorov
authors.forename: R
authors.initials: R
authors.lastname: Grabherr
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D004285:Dogs; D005814:Genes, Viral; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D009981:Influenza B virus / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*