The most commonly used baculovirus for foreign gene expression is Autographa californica multiple nucleopolyhedrovirus (AcMNPV)2,3. AcMNPV has a circular, double-stranded, super-coiled DNA genome (133894 bp; Accession: NC_001623)4 packaged in a rod-shaped nucleocapsid. AcMNPV has a bi-phasic life cycle resulting in the production of two virus phenotypes, a budded virus (BV) and an occluded virus (OB). BVs contain single, rod-shaped nucleocapsids enclosed by an envelope containing a membrane-fusion protein (GP64), which is acquired by budding through the infected cell surface5. This form of the virus is much more infective for cultured insect cells6, compared to the OB phenotype and is responsible for cell-to-cell transmission in the early stages of infection7. In the late stages of infection large numbers of OBs are formed. These consist of multiple rod shaped nucleocapsids enclosed by an envelope acquired de novo and embedded within polyhedra. The major component of polyhedra is polyhedrin (polh)8,9 which is produced by the powerful transcriptional activity of the polh promoter. OBs protect the virus and allow them to survive between hosts, within the environment.

However, the polh gene is non-essential for virus replication in insect cells3 and this has lead to the development of the widely-used baculovirus expression vector system-summers-,. Initially, homologous recombination between a transfer vector containing the gene of interest and the polh locus resulted in recombinant viruses, which could only be identified by visual inspection of plaques for the absence of polyhedra. The frequency of recombination using this system was very low (1%)12 and recombinant virus plaques were often obscured by parental virus plaques. This problem was partially addressed by the insertion of the E. coli LacZ gene into the virus genome in addition to the gene of interest. The recombinant virus plaques could then be stained blue by the addition of X-gal (5-bromo-4-chloro-3-indolyl ß-D-galactopranoside) against a background of colourless parent plaques. However, this did not improve the low recombination efficiency and resulted in the contamination of recombinant protein with ß-galactosidase.

The recombination efficiency was improved by the addition of a unique restriction enzyme site (Bsu36I) at the polyhedrin locus (AcRP6-SC)13. Linearization of the virus genome reduced the infectivity of the DNA but increased the proportion of recombinant virus produced to 30%. A double recombination event between the transfer vector and the linear DNA re-circularised the virus genome, restoring infectivity. LacZ was then introduced at the polh locus, producing AcRP23.lacZ. A Bsu36I restriction site within LacZ allowed for the selection of colourless plaques against a blue parental background in the presence of X-gal13. Almost 100% recombination efficiency was achieved by further modifications to produce BacPAK610,11. BacPAK6 contains an E. coli lacZ insert at the polh locus and Bsu36I restriction enzyme sites in the two flanking genes on either side of lacZ. Digestion with Bsu36I removes the lacZ insert and a fragment of an essential gene (ORF 1629) producing linear virus DNA (BacPAK6) that is unable to replicate within insect cells. Co-transfection of BacPAK6 with a transfer vector containing the gene of interest under the control of the polyhedrin promoter (or other baculovirus or non-baculovirus promoter) flanked by baculovirus sequences homologous to those removed by Bsu36I digestion, restores ORF1629 and re-circularises the virus by allelic replacement. The recombinant baculovirus DNA is then able to replicate in insect cells and in the late phase of infection, virions are assembled and recombinant baculoviruses are produced.

However, Bsu36I digestion is never 100% efficient and the final virus population will always contain a mixture of recombinant and parental virus. This requires the isolation of recombinants from parental background by several rounds of plaque purification, a time-consuming and labour-intensive process.

The new flashBAC system dispenses with this problem.
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