Figure 2

Selective removal of marker genes. A) Cartoon showing strategy for Recombineering of an expression cassette (expression) into the AcMNPV Bacmid DNA and selective removal of only the marker genes (selection) by Cre mediated recombination. B) PCR using primers labelled a and b in A using: Lanes 1-2, two independent bacmid recombinants following red recombination; Lanes 3-6, four independent recombinants following Cre mediated recombination to remove the bacterial selectable markers; Lane 7, no template; Lane 8, unmodified bacmid DNA template; Lane 9, plasmid DNA template containing the selectable marker cassette; Lane 10, DNA marker. PCR products corresponding to the sizes predicted for the parental and recombinant products of the Cre mediated recombination are labelled P and R respectively. C) Sequencing trace file of a representative recombinant from the PCR analysis in B confirming the presence of a defective lox P incorporating the loxP71 and loxP66 arms that render the recombinant incapable of undergoing further Cre-mediated recombination. D) Renilla luciferase activity of the parental and recombinant bacmids from B when transfected into insect cells. Renilla luciferase activity was assayed at 48 hours post infection after on passage 2 of the recombinant virus. Background activity from uninfected cells is labelled Cells.