Download Making recombinant baculovirus

Generation of recombinant baculovirus using Baculo Gold protocol (BD Pharmingen)
N. B. You’ll need the Baculo Gold kit (BD Pharmingen cat no 21100K, 5 transfections
$365)
1. Plate the cells to be transfected (either Sf9 or Sf21 insect cells) either the same day or
the day before at 2-3 x 106 in a T25 flask (don’t use p60 dish, medium will
evaporate).
The cells should be 50-70 % confluent at the time of transfection. Grow
the cells in Grace’s supplemented medium (Gibco 11605-094) further supplement ed
with Gentamicin (Gibco 15710-064, 2.5 ml per 500 ml medium) and 10 % fetal calf
serum.
2. Use a Falcon 2054 tube to mix the following: 0.5 µg BaculoGold DNA (baculovirus
genomic DNA digested with certain restriction enzyme to generate a lethal deletion,
which can only be rescued upon homologous recombination with the transfer vector).
Add 2-5 µg of the recombinant baculovirus transfer vector (e.g. gene of interest
cloned into pVL1392/93, orientation must be correct relative to the polyhedrin
promoter, some vectors (pFastBac) are not compatible with this system) and pipet to
mix thoroughly.
3. Add 1 ml of transfection buffer B.
4. Aspirate old medium from the cells that are to be transfected, and replace with 1 ml
of transfection buffer A.
5. Add the 1 ml transfection buffer B/ DNA solution dropwise to the transfection buffer
A in the T25 flask, gently swirling after every 3-5 drops. This is most conveniently
done by temporarily tilting the flask so all the transfection buffer A is at the bottom of
the flask.
6. A very fine (milky white) calcium phosphate precipitate should form. Note that the
solutions barely cover the cells (the cells should not dry out). Incubate at 27 ˚C for 4
hrs
7. Carefully aspirate the transfection mix and replace with 4 ml of complete medium.
8. After 4-5 days (usually 5) the supernatant can be harvested and saved. Spin it down
to get rid of any cells in the medium and transfer to a new tube. Store at 4 ˚C
protected from light. NB the recombinant baculovirus should at this point have a
very low titer and needs to be amplified at least twice.
9. For amplification of virus stock do the following: Seed the cells in a T75 flask so
they’re approximately 70 % confluent. Aspirate the medium, and replace with 2 ml
of virus stock from the original transfection + 2 ml of complete medium. Incubate on
a rocker table at room temperature for 1 hr; aspirate virus and replace with 20 ml of
complete medium. At 4-5 days post infection (5 usually works best) harvest the
supernantant containing the virus. Spin down to remove cells and transfer virus to a
new tube (store at 4 ˚C). Repeat the procedure. Waiting for 4-5 days allows multiple
rounds of virus infection releasing more and more virus into the supernatant. Hence,
titer should go up quite dramatically.