BacPAK6 linear DNA (cat#101101/101102/101103) is the original convenient, highly efficient reagent for generating recombinant viruses. It comprises a modified Autographa californica nucleopolyhedrovirus (AcMNPV) genome with lacZ inserted in place of the native polyhedrin gene coding region. Digestion of BacPAK6 DNA at three sites with Bsu36I removes lacZ and part of ORF 1629, which is an essential AcMNPV gene. The linear virus DNA is unable to initiate a replication cycle in insect cells. Co-transfection of insect cells with linear BacPAK6 DNA and a compatible transfer vector containing the complete ORF 1629 and a foreign gene of interest results in recombination and restoration of the circular, infectious virus genome. Over 95% of the virus derived from the co-transfection comprises recombinant virus. Usually, a single plaque assay titration incubated with X-gal and counterstained with neutral red is sufficient to differentiate parental blue from recombinant white (colourless) plaques. These can be readily isolated and amplified to working stocks of purified recombinant virus in a few days.
OET’s flashBAC™ range of expression vectors build upon the BacPAK6 technology but have removed the need to separate the parental and recombinant virus. As only recombinant virus is produced after co-transfection, the flashBAC™ expression system reduces the whole process to a simple one-step procedure.