(a) For each transfection sample, 0, 3, 6, 9, 12, and 15 μL of
DMRIE-C is added to a 1.5 mL microcentrifuge tube
containing 1 mL Opti-MEM I reduced-serum medium
at room temperature.
(b) In vitro–transcribed recombinant RNA (10 μL, ~5 μg)
and helper RNA (5 μL, ~2.5 μg) are mixed.
(c) The RNA (15 μL total) is added to each tube containing
the DMRIE and vortexed briefly.
4. The lipid-RNA complex is immediately added to the cells and
incubated at 37
C for 4 h.
5. The transfection medium is replaced with prewarmed (37
C)
complete BHK medium and the BHK-21 cells are incubated at
37
C overnight in an incubator with 5% CO 2 .
3.6 Harvest
of Recombinant SFV
Particles
Production of recombinant SFV particles occurs within the first
24 h, resulting in high titers of approximately 10
9 infectious particles/mL. The titers can be increased to some extent by extending
the incubation time by another 24 h.
1. Virus particles are harvested by carefully removing the medium
from the BHK-21 cells.
2. Virus stocks are sterilized and purified by forcing the harvested
medium through a 0.22 μm filter to remove cell debris and
possible contaminants.
3. Virus stocks are aliquoted before storage at À20
C (for weeks)
or at À80
C (for years) as repeated cycles of freezing and
thawing can reduce the titers significantly.
3.7 Activation
of Recombinant SFV
Particles
Although the conventional pSFV-helper vector has been initially
used, it is recommended to utilize the second-generation vector
pSFV-Helper2, as it contains three point mutations at the cleavage
site between the E2 and E3 proteins in the p62 precursor
[29]. Generation of infectious particles requires activation with
α-chymotrypsin as described below. In contrast to SFV particles
generated with the pSFV-helper vector, particles produced with the
pSFV-Helper2 vector are conditionally infectious and thereby prevent accumulation of replication-proficient particles through
recombination, thus providing an additional level of biosafety.
1. Particles are activated by addition of α-chymotrypsin at a final
concentration of 500 μg/mL for 20 min at room temperature.
2. The reaction is terminated by addition of aprotinin (trypsin
inhibitor) at a final concentration of 250 μg/mL and the
activated particles are ready to use.
72
Kenneth Lundstrom
DMRIE-C is added to a 1.5 mL microcentrifuge tube
containing 1 mL Opti-MEM I reduced-serum medium
at room temperature.
(b) In vitro–transcribed recombinant RNA (10 μL, ~5 μg)
and helper RNA (5 μL, ~2.5 μg) are mixed.
(c) The RNA (15 μL total) is added to each tube containing
the DMRIE and vortexed briefly.
4. The lipid-RNA complex is immediately added to the cells and
incubated at 37
C for 4 h.
5. The transfection medium is replaced with prewarmed (37
C)
complete BHK medium and the BHK-21 cells are incubated at
37
C overnight in an incubator with 5% CO 2 .
3.6 Harvest
of Recombinant SFV
Particles
Production of recombinant SFV particles occurs within the first
24 h, resulting in high titers of approximately 10
9 infectious particles/mL. The titers can be increased to some extent by extending
the incubation time by another 24 h.
1. Virus particles are harvested by carefully removing the medium
from the BHK-21 cells.
2. Virus stocks are sterilized and purified by forcing the harvested
medium through a 0.22 μm filter to remove cell debris and
possible contaminants.
3. Virus stocks are aliquoted before storage at À20
C (for weeks)
or at À80
C (for years) as repeated cycles of freezing and
thawing can reduce the titers significantly.
3.7 Activation
of Recombinant SFV
Particles
Although the conventional pSFV-helper vector has been initially
used, it is recommended to utilize the second-generation vector
pSFV-Helper2, as it contains three point mutations at the cleavage
site between the E2 and E3 proteins in the p62 precursor
[29]. Generation of infectious particles requires activation with
α-chymotrypsin as described below. In contrast to SFV particles
generated with the pSFV-helper vector, particles produced with the
pSFV-Helper2 vector are conditionally infectious and thereby prevent accumulation of replication-proficient particles through
recombination, thus providing an additional level of biosafety.
1. Particles are activated by addition of α-chymotrypsin at a final
concentration of 500 μg/mL for 20 min at room temperature.
2. The reaction is terminated by addition of aprotinin (trypsin
inhibitor) at a final concentration of 250 μg/mL and the
activated particles are ready to use.
72
Kenneth Lundstrom