In the baculovirus shuttle vector (bacmid) system, a helper plasmid and a donor plasmid are
employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in
Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed
bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,
and hence to the production of defective virions. In this study, to facilitate the preparation of
plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing
their replication origins with that of a temperature-sensitive (ts) plasmid, pSIM6. Using the resulting
ts helper plasmid pMON7124ts and the ts donor plasmid pFB1ts-PH-GFP, a recombinant bacmid,
bAcWT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The
plasmids were then removed by culturing at 37 °C. For bAcWT-PG(-), the infectious progeny virus
titer and the protein expression level under the control of the polyhedrin promoter were similar to
those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will
be useful for obtaining plasmid-free bacmids for both heterologous protein production and
fundamental studies of baculovirus biology.