flashBAC Manual - Oxford Expression Technologies

addition to the gene of interest. The recombinant virus plaquescould then be stained blue by the addition of X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopranoside) against a background ofcolourless parent plaques. However, this did not improve thelow recombination efficiency and resulted in the contaminationof recombinant protein with β-galactosidase.The efficiency with which recombinant virus could be recoveredwas improved by the addition of a unique restriction enzymesite (Bsu36I) at the polyhedrin locus (AcRP6-SC). Linearizationof the virus genome prior to homologous recombinationreduced the infectivity of the virus DNA but increased theproportion of recombinant virus recovered to 30%.Homologous recombination between the transfer vector and thelinear DNA re-circularised the virus genome, restoring infectivityand the production of virus particles. LacZ was then introducedat the polyhedrin gene locus, replacing the polyhedrin codingregion, producing AcRP23.lacZ. A Bsu36I restriction site withinlacZ allowed for more efficient restriction of the linear DNA priorto homologous recombination and the presence of lacZ allowedthe selection of colourless recombinant virus plaques against abackground of blue parental virus plaques in the presence of X-gal 11 .Greater than 90% recovery of recombinant virus plaques wasachieved by further modifications to produce BacPAK6 12 .BacPAK6 contains the E. coli lacZ gene inserted at thepolyhedrin gene locus and Bsu36I restriction enzyme sites intwo flanking genes on either side of lacZ. Digestion withBsu36I removes the lacZ gene and a fragment of an essentialgene (ORF 1629) 10 producing linear virus DNA (BacPAK6) thatis unable to replicate within insect cells. Co-transfection ofinsect cells with BacPAK6 DNA and a transfer vector containingOXFORD EXPRESSION TECHNOLOGIES 14