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MAR TIN MARX
Subprotocol 7
lnfection of \j!AM Packaging Cells
Procerlure
1. Growup the \j/AM cells in 75 cm 2 tissue culture flasks containing cDMEM
supplemented with penicillin, Streptomycin and 10% bovine calf serum.
Again, be sure not to use fetal calf serum because this will detach the
\j!AM cells immediately! You will need two flasks of cells: one for the
infection, the other for the monitaring of the selection process.
2. Grow until the cells are about 75-90% confluent.
3. Thaw a cryo tube containing 5 ml of ecotropic viral particles (supernatants from transfected \j/2 cells). Add polybrene to a final concentration
of 8 J..tg/ml; this will make the viruses sticky. Filter through a 0.45 J..tm
filter. Remave old medium by aspiration and replace with the viral
stock-polybrene mixture. Return the cells to the incubator and let the
viruses act overnight.
4. Remave the viral stock by aspiration and replace with regular growth
medium.
5. After 24 hours, start selection with G418 in the same way as described
above.
6. When the control cells are completely destroyed and detached, start harvesting the amphotropic viral particles as described above for the ecotropic viruses.
Subprotocol 8
lnfection of Target Cells
Procerlure
The procedure is exactly the same as described for the generation of amphotropic virus stocks. W e usually incubate subconfluent target cells with
the virus overnight. However, infections may be achieved in one hour. Short
incubation times may be preferable if the medium used during the virus
harvest (cDMEM with 10% bovine calf serum) does not meet the growth
MAR TIN MARX
Subprotocol 7
lnfection of \j!AM Packaging Cells
Procerlure
1. Growup the \j/AM cells in 75 cm 2 tissue culture flasks containing cDMEM
supplemented with penicillin, Streptomycin and 10% bovine calf serum.
Again, be sure not to use fetal calf serum because this will detach the
\j!AM cells immediately! You will need two flasks of cells: one for the
infection, the other for the monitaring of the selection process.
2. Grow until the cells are about 75-90% confluent.
3. Thaw a cryo tube containing 5 ml of ecotropic viral particles (supernatants from transfected \j/2 cells). Add polybrene to a final concentration
of 8 J..tg/ml; this will make the viruses sticky. Filter through a 0.45 J..tm
filter. Remave old medium by aspiration and replace with the viral
stock-polybrene mixture. Return the cells to the incubator and let the
viruses act overnight.
4. Remave the viral stock by aspiration and replace with regular growth
medium.
5. After 24 hours, start selection with G418 in the same way as described
above.
6. When the control cells are completely destroyed and detached, start harvesting the amphotropic viral particles as described above for the ecotropic viruses.
Subprotocol 8
lnfection of Target Cells
Procerlure
The procedure is exactly the same as described for the generation of amphotropic virus stocks. W e usually incubate subconfluent target cells with
the virus overnight. However, infections may be achieved in one hour. Short
incubation times may be preferable if the medium used during the virus
harvest (cDMEM with 10% bovine calf serum) does not meet the growth