flashBAC Manual - Oxford Expression Technologies

7 Introduction to the Baculovirus ExpressionSystem and flashBAC Technology7.1 BaculovirusesBaculoviruses are insect viruses, predominantly infecting insectlarvae of the order Lepidoptera (butterflies and moths) 1 . Abaculovirus expression vector is a recombinant baculovirus thathas been genetically modified to contain a foreign gene ofinterest, which can then be expressed in insect cells undercontrol of a baculovirus gene promoter. The most commonlyused baculovirus for foreign gene expression is Autographacalifornica nucleopolyhedrovirus (AcMNPV) 2,3 . AcMNPV has acircular, double-stranded, super-coiled DNA genome (133894bp; Accession: NC_001623) 4 , packaged in a rod-shapednucleocapsid. The nucleocapsid can be extended lengthwaysand thus the DNA genome can accommodate quite largeinsertions of foreign DNA. The AcMNPV genome forms thebasis of the flashBAC DNA provided in this kit.AcMNPV has a bi-phasic life cycle resulting in the production oftwo virus phenotypes: budded virus (BV) and occlusion-derivedvirus (ODV). BVs contain single, rod-shaped nucleocapsidsenclosed by an envelope (Figure 1) containing a membranefusionprotein (GP64). GP64 is acquired when thenucleocapsids bud through the host cell plasma membrane 5 .The BV form of the virus is 1000-fold more infectious forcultured insect cells 6 , compared to the ODV phenotype, and isresponsible for cell-to-cell transmission in the early stages ofinfection 7 . It is the BV form of the virus that delivers the foreigngene into the host insect cell.OXFORD EXPRESSION TECHNOLOGIES 10