Cloning of complete genomes of large dsDNA viruses by in vitro transposition of an F factor containing transposon

An improved bacmid technology for cloning complete genomes of large dsDNA viruses with circular genomes has been developed and tested. The system, termed EZ::BAC, is based on Escherichia coli F factor replicon, a chloramphenicol resistant marker gene with the mosaic ends recognized specifically by the transposase of the Tn5. In vitro transposition was carried out for the baculovirus shuttle vector pMON14272 (136 kb) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome (134 kb) as target DNAs. Transposon EZ::BAC was inserted randomly into the target DNAs, leading to 9 bp duplication of the flanking end at the insertion site. One of the obtained AcMNPV::BACs replicated in Sf21 cells after transfection. The random in vitro generation of viral bacmids using EZ::BAC facilitates the host-independent propagation of intact and functional viral genomes in E. coli cells and does not require sequence information of the target DNA as is necessary for the generation of bacmids in conventional systems.