A novel system for the generation of baculoviruses mutant for an essential gene.

Abstract

Currently, to delete an essential gene from a baculovirus genome, a cell line stably expressing the gene to be knocked-out should be first generated, which is time-consuming. Alternatively, essential genes can be deleted in E. coli using the λ Red recombination system, which requires an electroporation system. Here, based on homologous recombination in insect cells, we develop an alternative efficient system that requires neither generation of a cell line nor an electroporation system. Using puc19-based inverse PCR, a transfer vector for deleting BmNPV orf92 (Bm92, an essential gene) was efficiently constructed. A copy of Bm92 was introduced into the polyhedrin locus of BmNPV bacmid. The transfer vector was then co-transfected into BmN cell with the modified bacmid to enable homologous recombination at the Bm92 locus. An agarose-free approach was developed for the purification of Bm92-disrupted bacmid viruses in insect cells. Subsequently, BmN cells were co-infected with purified Bm92-disrupted bacmid viruses and unmodified bacmid viruses to allow recombination at the Tn7 insertion site between the two viruses. Finally, bacmid DNA extracted from BmN cells was transformed into chemically-treated competent DH10B cells, and blue colonies containing Bm92-disrupted bacmid were selected using PCR. For its efficiency and convenience, the system has great potential to be used for the generation of baculovirus knockout mutants.