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were cloned into pIEx/Bac-1 transfer vector (Novagen, EMD-Millipore) for
recombination in insect cells after cotransfection with AcNPV BacMagic DNA
(Novagen). Alternatively, cDNAs for N- or C-terminally His
6
-StrepII-tagged
recombinant proteins were subcloned into Invitrogen's pFastBac1 transfer plasmid.
The latter technique, which was more complicated and time-intensive but more
cost-effective, is expounded upon here.
After selection and confirmation of recombinant pFastBac1 plasmids, recombi-
nation was performed in
Escherichia coli
containing a bacmid vector that encodes all
baculovirus production genes. This recombination was performed in 20
m
lof
DH10Bac
E. coli
, which contains the appropriate helper plasmids and parental bac-
mid. DH10Bac
E. coli
are shipped in 100
m
l one-shot-use tubes, but we found that
much less is required for transformation and selection of recombinant nonparental
colonies. For specifics on the transformation protocol, refer to the DH10Bac
E. coli
online user manuals (Invitrogen). We noted that 100
m
l of a 1:100 dilution of trans-
formation reactions (into sterile LB) after 4 h of growth for transformation recovery
was ideal for selecting distinctly separated colonies on 10 cm LB-agar Petri dishes
containing 50
m
g/ml kanamycin, 7
m
g/ml gentamicin, 10
m
g/ml tetracycline,
100
m
g/ml BluoGal, and 40
m
g/ml. After a 48 h transformation of the DH10Bac
E. coli
with 1
m
l of purified recombinant pFastBac1 plasmid, white colonies were
selected using the aforementioned three-antibiotic-BluoGal-IPTG selection system.
The reason that white colonies are desired and used for selection is because the
parental bacmid contains DNA encoding a
LacZ
a
peptide in between recombination
sites that is removed when recombination occurs with pFastBac1; hence recombinant
colonies are white, unlike parental bacmids which are blue.
The DNA flanking this region is also used for PCR-based confirmation of recom-
bination. In order to confirm the identity of a recombinant bacmid, colonies were
replica plated and subjected to PCR using Invitrogen-recommended primers that
flanked the recombination sites: pUC/M13 forward (5
0
-CCCAGTCACGACGTTG-
TAAAACG-3
0
) and pUC/M13 reverse (5
0
-AGCGGATAACAATTTCACACAGG-
3
0
). A recombinant empty bacmid's PCR product was
300 bp, while a bacmid
transposed with pFastBac1 yielded a PCR product of
the size of the
cDNA that was utilized. We experimented with shortening the duration of the
PCR using one internal primer and one of the recommended flanking primers.
Appropriately selected colonies were used to inoculate LB medium containing
kanamycin, gentamicin, and tetracycline, and bacmids were purified by high-
molecular weight (MW) plasmid precipitation (refer to
Section 2.1.4
). Recombinant
bacmid DNA (2
m
g) was then carefully pipetted for transfection into monolayers of
0.5-1
2300 bp
þ
10
6
Sf9 cells in six-well plates using Cellfectin II liposomal reagent accord-
ing to the manufacturer's instructions (Invitrogen). Media was replaced after 3-5 h
of transfection as recommended by Invitrogen. After 4-5 days of incubation, we
observed maximal signs of viral infection—increased cell diameter and nuclear size,
cessation of cell growth, granular appearance, and cell detachment from the culture
dishes. At this time, P1 generation baculovirus supernatant was harvested by low-
speed centrifugation at 500
g
and used for 100-fold titer amplification in two steps
