An efficient way to introduce unique restriction endonuclease sites into a baculovirus genome

Recombinant baculoviruses which can be linearized at unique sites with restriction endonucleases can greatly facilitate the construction of other recombinants including baculovirus expression vectors and site-specific mutants. We designed a strategy to introduce unique restriction endonuclease sites at virtually any location in a baculovirus genome. The unique sites were first introduced onto a transfer plasmid which also contained in the vector portion of the plasmid an E. coli lacZ gene and a Sse8387I site, a sequence which is not found in the viral genome. Cotransfection of the transfer plasmid and circular viral DNA generated single-crossover recombinant viruses which could be distinguished as blue plaques in the presence of X-gal, a chromogenic indicator for lacZ. Single-crossover recombinants were purposefully isolated and propagated to generate double-crossover recombinants. Viral DNA isolated from the mixed virus population was digested with Sse8387I to linearize only the single-crossover viral DNA; double-crossover recombinants in the progeny viral population resulting from transfection with the Sse8387I-linearized viral DNA mixture were thus highly enriched, making the task of screening much easier. To demonstrate the feasibility of this approach, we introduced Bsu36I sites into the orf24 and the vlf-1 regions of Autographa californica multiple-nucleocapsid nuclear polyhedrosis virus (AcMNPV) to generate recombinant viruses vncBsuorf24 and vncBsuvlf1, respectively. Both recombinant viruses were obtained by screening only ten plaques. This method should also be applicable to other kinds of mutations and may be applicable to other double-stranded DNA viruses.