22 Expression in Eukaryotic Cells: Gene Transfer Using Retroviral Vectors
347
1. Prepare a stock solution of geneticin (16 mg/ml in PBS), fllter through a
sterile 0.2 f..!m filter, store at -20°C. Be aware that the geneticin powder
consists only of about 50-65% active substance, the exact proportion is
given on the label. Your stock solution should be 16 mg!ml of the active
substance.
2. Y ou may begin selection 24-48 hours after transfection. The cells should
be confluent at this time point.
3. Add G418 to complete medium to a final concentration of 400 f...l.g/ml.
4. Remove old medium by aspiration and replace with G418 medium. Do
not forget to treat the control cells not exposed to the virus with G418.
After 1 or 2 days the non-resistant cells start to round up and detach. Feed
the cells twice or three times per week with fresh medium supplemented
with G418. After 5-10 days, all cells in the control flask should be detached and dead. At this time point outgrowth of succesfully transfected
\j/2 cells should be easily visible.
5. Growthe cells until confluency, then splitin a 1:2-1:4 ratio, depending on
their growth capacity. About two weeks after transfection the selection
medium is replaced with normal medium.
Harvesting of ecotropic replication incompetent viruses
Wait until the cells are subconfluent, remove old medium by aspiration and
cover the celllayer with 5 ml of fresh medium. Be sure not to use medium
supplemented with geneticin! Do not use larger volumes of medium to
achieve higher concentrations of viral particles. After 24 hours, remove
the medium, filter through a 0.45 f..!m filter to remove cellular debris ( do
not use 0.2 f..!m membranes to avoid loss of viruses!) and store in a cryo
tube at -70°C. Subsequently, the viruses may be harvested a second time
in the same way. Afterwards, the monolayer will be confluent and the cells
should be passaged at a 1:2-1:4 ratio. When theyreach subconfluency, a new
harvesting cycle may start. The viral stocks may be stored at -70°C indefinitely. The transfected \j/2 cells can be stored in liquid nitrogen for future
use.
347
1. Prepare a stock solution of geneticin (16 mg/ml in PBS), fllter through a
sterile 0.2 f..!m filter, store at -20°C. Be aware that the geneticin powder
consists only of about 50-65% active substance, the exact proportion is
given on the label. Your stock solution should be 16 mg!ml of the active
substance.
2. Y ou may begin selection 24-48 hours after transfection. The cells should
be confluent at this time point.
3. Add G418 to complete medium to a final concentration of 400 f...l.g/ml.
4. Remove old medium by aspiration and replace with G418 medium. Do
not forget to treat the control cells not exposed to the virus with G418.
After 1 or 2 days the non-resistant cells start to round up and detach. Feed
the cells twice or three times per week with fresh medium supplemented
with G418. After 5-10 days, all cells in the control flask should be detached and dead. At this time point outgrowth of succesfully transfected
\j/2 cells should be easily visible.
5. Growthe cells until confluency, then splitin a 1:2-1:4 ratio, depending on
their growth capacity. About two weeks after transfection the selection
medium is replaced with normal medium.
Harvesting of ecotropic replication incompetent viruses
Wait until the cells are subconfluent, remove old medium by aspiration and
cover the celllayer with 5 ml of fresh medium. Be sure not to use medium
supplemented with geneticin! Do not use larger volumes of medium to
achieve higher concentrations of viral particles. After 24 hours, remove
the medium, filter through a 0.45 f..!m filter to remove cellular debris ( do
not use 0.2 f..!m membranes to avoid loss of viruses!) and store in a cryo
tube at -70°C. Subsequently, the viruses may be harvested a second time
in the same way. Afterwards, the monolayer will be confluent and the cells
should be passaged at a 1:2-1:4 ratio. When theyreach subconfluency, a new
harvesting cycle may start. The viral stocks may be stored at -70°C indefinitely. The transfected \j/2 cells can be stored in liquid nitrogen for future
use.