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High titer recombinant virus stocks (~ 2 x 108 pfu/ml) are
used for infection of cells at optimal Multiplicity Of Infection
(MOI = # of virus / # of cells) resulting in maximum protein production.
Prepare large stocks of virus by infecting insect cells at a low multiplicity
of infection (MOI <1) and harvesting supernatant 3-5 days post infection
(pi). It is critical to use a low MOI because passaging the virus at high
MOI increases the possibility of virus with mutations in their genome.
The number of mutant virus is also increased by serial passage; it may
be advantageous to maintain a low-passage seed stock from which larger
working stocks are amplified. Eventually, the titer in the seed stock
will be reduced through storage, and it becomes necessary to generate
a new passage seed stock.
Since recombinant virus generated using BD BaculoGold™ are greater
than 99% of the total virus population, it may not be necessary to initially
prepare all stocks from a clonal viral population. However, if there is
a reduction in protein production after multiple passages of a viral stock,
it may be necessary to isolate clonal viral populations by end-point dilution
assay (EPDA) or plaque purification. After verification of protein production,
the clonal virus population can be amplified to produce a high titer stock.
The viral stock is then ready for large scale protein production.
Protocol
- Seed 2 x 107 Sf9 cells on a 15 cm plate (BD Falcon™,
Cat. No. 352097). Allow cells to attach for 15 min, confirm ~ 50% confluency
by visualization using a light microscope. Change to fresh TNM-FH medium.
- Add 100 µl -1 ml of your low titer recombinant stock to the
plate. If you know the virus titer of your stock solution, use MOI <1.
Repetitive infections with an MOI > 1 may select for mutants which no
longer express your gene.
- Incubate the cells at 27°C for 3 days. Check for signs of infection
2 days after the start of infection.
- Harvest the supernatant from the plate. Remove cellular debris by
centrifugation at 10,000 x g.
- Store the virus supernatant in a sterile tube at 4°C for up to
six months. For longer storage periods, virus supernatant should be
frozen in aliquots at -80°C.
- Determine the virus titer of your amplification solution using the
plaque assay procedure or the modified end-point dilution assay. Amplification
typically is done 2 or 3 times to attain a high viral titer (~2 x 108
/ml).
Storing Virus Particles
Supernatants containing baculovirus particles may be stored in a sterile
tube at 4°C for up to 6 months or frozen, in aliquots, at -80°C
for a longer period of time. If frozen, avoid multiple freeze and thaw
cycles. Upon freezing, the viral titer may decrease and should be re-amplified
when thawed. Store viral stocks in the dark; titers appear to decrease
when exposed to fluorescent light for prolonged periods of time. The best
way to preserve a recombinant virus is to isolate its DNA and store it
at -80°C.
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