High-throughput baculovirus expression in insect cells

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 824; p. 609
• Main Authors: Hitchman, Richard B, Possee, Robert D, King, Linda A
• Format: Journal Article
• Language: English
• Published: United States 2012
• Subjects: Animals; Baculoviridae - genetics; Baculoviridae - metabolism; Biotechnology - methods; Blotting, Western; Cell Culture Techniques - methods; Cell Line; DNA Primers - genetics; Electrophoresis, Polyacrylamide Gel; Genetic Vectors - genetics; Homologous Recombination - genetics; Plasmids - genetics; Polymerase Chain Reaction - methods; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spodoptera
• ISSN: 1940-6029
• DOI: 10.1007/978-1-61779-433-9_33

Historically, it has been proved difficult to adapt the traditional baculovirus expression systems to an automated platform because of the complexity of the processes involved. One of the major bottlenecks is the selection of recombinant from parental viruses. We have developed a bacmid vector (flashBAC™) that does not require any form of selection pressure to separate recombinant virus from nonrecombinant parental virus. The method relies on homologous recombination in insect cells between a transfer plasmid containing the gene of interest and a replication-deficient bacmid. The gene of interest replaces the bacterial replicon at the polyhedrin locus, simultaneously restoring a virus gene essential for replication, and as only recombinant virus can replicate, no further separation techniques are required. This chapter describes methods for producing and expression testing multiple recombinant baculoviruses on automated platforms using the flashBAC system.