To replace the BV polyhedrin promoter with the cytomegalovirus
(CMV) promoter, a CMV promoter fragment
(
BglII-
XhoI) from the pcDNA3.1 vector
(Invitrogen, Carlsbad, CA) was cloned into pFastBac1 (Life
Technologies, Inc., Rockville, MD) between the sites
SnaBI
and
BamHI (vector pFastCMV). The coding region for the
enhanced green fluorescent protein (GFP) was then transferred as a
fragment
EcoRI-
NotI from pEGFP1 (Clontech
Laboratories, Palo Alto, CA) into pFastCMV opened at the
EcoRI and
NotI sites. Similarly, the coding
sequence for either the blue fluorescent protein (BFP) or the red
fluorescent protein (RFP) was transferred as a fragment
EcoRI-
NotI from pEBFP-N1 (Clontech Laboratories)
or pDsRed1-N1 (Clontech Laboratories), respectively, into pFastCMV,
opened at the
EcoRI and
NotI sites. The
expression cassette was then transferred into the BV shuttle vector by
transposition. SF9 insect cells were transfected with the recombinant
bacmid using cationic liposome–mediated transfection (CellFECTIN
reagent; Life Technologies, Inc.). After amplification of the rBV for 3
days in SF9 cells, cell debris was removed by centrifugation at
1000
g for 15 minutes, and the rBV-CMV-GFP was then
concentrated from the cell culture supernatant by centrifugation at
80,000
g for 1 hour at 4°C. The resultant pellet containing
the rBV was resuspended in PBS (1.5 mM
KH
2PO
4, 12 mM
NaH
2PO
4, 2.7 mM KCl, and
139 mM NaCl [pH 7.4]) and used for in vitro and in vivo experiments.
The rBV was further purified by the 25% to 60% linear sucrose
gradient centrifugation method (96,000
g for 3
hours).
34 A white band containing the rBV formed at
approximately 47% sucrose, was collected and diluted approximately 10
times in PBS. The rBV was collected by centrifugation at
80,000
g for 75 minutes, resuspended in 200 μl PBS, and
then used for the experiments. The rBV titer was determined using a
rapid titer kit (BacPak BV; Clontech).