264
1. Day 1: For lentiviral vector production complement 10 ml of
Advanced DMEM with 2% (v/v) FCS and penicillin (100 U/
ml)/streptomycin (100 μg/ml). Further add 20 μl cholesterol,
20 μl egg lecithin, and 1× chemical defined lipid concentrate.
Dilute HEK293T cells in 10 ml with a density of 0.75 × 10
5
cells/cm
2
and seed them in a 10-cm plate.
For retroviral vector production dilute Platinum E cells
with a density of 0.75 × 10 cells/cm
2
in 10 ml culture medium
and seed them in a 10-cm plate (see Note 8).
2. Day 2: Cells should have reached ~60–70% confluence before
continuing. For lentiviral vector production prepare PEI transfection mix by adding 5 μg pLTR-G, 5 μg pCD/NL BH*DDD,
10 μg pWM043 (or pWM023), and 1 ml opti-MEM. Afterward,
add 60 μl PEI and vigorously vortex the solution for 5 s. For
retroviral vector production dilute 8 μg PA-AKT (or pWM029)
in 1 ml opti-MEM. Then add 24 μl PEI and vortex vigorously.
In both cases, incubate the mixture for 15 min at room
temperature before adding it dropwise on the cells. Gently
rock the plate to ensure an even distribution of transfection
mix. Place the cells back into the incubator.
3. After 5 h replace the transfection medium with fresh Advanced
DMEM including the above-listed additives and return the
cells back into the incubator. At this time point the medium
may already contain viral particles. BSL2 precautionary measures have to be undertaken when working with lentiviral
vectors.
4. Day 3: As target cells seed MCF7 cells, for lentiviral vector
transduction, or C2C12 cells, for retroviral vector transduction, in 6-well plate with a density of 10
5
and 0.3 × 10
5
cells/
cm
2
, respectively, according to the procedure described in
Subheading 3.1.
5. Day 4: After 48 h incubation the transfected HEK293T/
Platinum E cells have produced a sufficient amount of viral
particles. Collect the supernatant, which contains either the
retro- or lentiviral particles, using a serological pipette and filter
it through a 0.45 μm sterile filter into a 50 ml conical centrifuge tube (see Note 9). Add polybrene solution to a final concentration of 8 μg/ml to the viral particle-suspension.
6. Replace the medium of the target cells with a total of 2 ml viral
particle-suspension. For a double transduction use 1 ml of each
viral particle-suspension (e.g., 1 ml of pWM043 and pWM023
harboring vectors). Next place cells back into the incubator for
15 min. After incubation centrifuge plates for 30 min at 1100 × g
(see Note 10). Subsequently to centrifugation discard viral
particle containing medium and add fresh culture medium
before returning cells into the incubator again.
3.1.2 Stable
Transduction
of Mammalian Cells
(See Note 7)
Wignand W.D. Mühlhäuser et al.
1. Day 1: For lentiviral vector production complement 10 ml of
Advanced DMEM with 2% (v/v) FCS and penicillin (100 U/
ml)/streptomycin (100 μg/ml). Further add 20 μl cholesterol,
20 μl egg lecithin, and 1× chemical defined lipid concentrate.
Dilute HEK293T cells in 10 ml with a density of 0.75 × 10
5
cells/cm
2
and seed them in a 10-cm plate.
For retroviral vector production dilute Platinum E cells
with a density of 0.75 × 10 cells/cm
2
in 10 ml culture medium
and seed them in a 10-cm plate (see Note 8).
2. Day 2: Cells should have reached ~60–70% confluence before
continuing. For lentiviral vector production prepare PEI transfection mix by adding 5 μg pLTR-G, 5 μg pCD/NL BH*DDD,
10 μg pWM043 (or pWM023), and 1 ml opti-MEM. Afterward,
add 60 μl PEI and vigorously vortex the solution for 5 s. For
retroviral vector production dilute 8 μg PA-AKT (or pWM029)
in 1 ml opti-MEM. Then add 24 μl PEI and vortex vigorously.
In both cases, incubate the mixture for 15 min at room
temperature before adding it dropwise on the cells. Gently
rock the plate to ensure an even distribution of transfection
mix. Place the cells back into the incubator.
3. After 5 h replace the transfection medium with fresh Advanced
DMEM including the above-listed additives and return the
cells back into the incubator. At this time point the medium
may already contain viral particles. BSL2 precautionary measures have to be undertaken when working with lentiviral
vectors.
4. Day 3: As target cells seed MCF7 cells, for lentiviral vector
transduction, or C2C12 cells, for retroviral vector transduction, in 6-well plate with a density of 10
5
and 0.3 × 10
5
cells/
cm
2
, respectively, according to the procedure described in
Subheading 3.1.
5. Day 4: After 48 h incubation the transfected HEK293T/
Platinum E cells have produced a sufficient amount of viral
particles. Collect the supernatant, which contains either the
retro- or lentiviral particles, using a serological pipette and filter
it through a 0.45 μm sterile filter into a 50 ml conical centrifuge tube (see Note 9). Add polybrene solution to a final concentration of 8 μg/ml to the viral particle-suspension.
6. Replace the medium of the target cells with a total of 2 ml viral
particle-suspension. For a double transduction use 1 ml of each
viral particle-suspension (e.g., 1 ml of pWM043 and pWM023
harboring vectors). Next place cells back into the incubator for
15 min. After incubation centrifuge plates for 30 min at 1100 × g
(see Note 10). Subsequently to centrifugation discard viral
particle containing medium and add fresh culture medium
before returning cells into the incubator again.
3.1.2 Stable
Transduction
of Mammalian Cells
(See Note 7)
Wignand W.D. Mühlhäuser et al.