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MARTIN MARX
Subprotocol 6
Transfeetion of \j/2 Packaging Cells
\j/2 (13) and \j/AM (14) cells are available from ATCC (American Type Culture Collection, Rockville, MD) or from laboratories that routinely work
with retroviral vectors. These cells originate from 3T3 fibroblasts and
arealready transfected with the coding sequences for the viral envelope proteins. \j/2 cells produce ecotropic viral particles that are only capable to infect rodent cells. \j/AM cells produce amphotropic viral particles that are
able to infect a broad range of mammalian cells, including those ofhuman
origin. The potential biohazard due to the possible generation of replication
competent viruses has stimulated the design of new packaging celllines that
eliminate the recombination events leading to helper virus production.
With the ecotropic packaging celllines \j/CRE (15) and GP+E86 (16) helper
virus rescue has never been reported. These packaging celllines should preferentially be used, when the target cells are of rodent origin. For the generattion of amphotropic viruses, the packaging cellline \j/CRIP {15) is probably a safe choice. The protocols described here for the first generation
packaging cells \j/2 and\j/ AM arealso applicable, when the new second generation packaging celllines are used. It is preferable to transfect the \j/2 cells
first and then use the ecotropic viral stocks to infect the \j/AM cells for the
production of amphotropic viral stocks. The reasons are:
• Infected cells, but not transfected cells, all produce infectious virus. Thus,
the highest titer virus stocks are produced from infected cells.
• Transfeetion of proviral DNAs apparently enhances the rate of recombination which Ieads to helper virus rescue, so transfection of the packaging cells which produce amphotropic virus (\j/AM, \j/CRIP and PA317)
should be avoided.
• \j/2 cells have a lower rate of helper-virus rescue than the \j/AM cells.
Therefore transfection of \j/2 carries only a small risk of generation of
replication-competent (helper) virus.
Note: For the transfection of \j/2 cells we usually use the DMSO shock method, but other methods such as lipofection, electroporation or calcium phosphate coprecipitation may be used.
MARTIN MARX
Subprotocol 6
Transfeetion of \j/2 Packaging Cells
\j/2 (13) and \j/AM (14) cells are available from ATCC (American Type Culture Collection, Rockville, MD) or from laboratories that routinely work
with retroviral vectors. These cells originate from 3T3 fibroblasts and
arealready transfected with the coding sequences for the viral envelope proteins. \j/2 cells produce ecotropic viral particles that are only capable to infect rodent cells. \j/AM cells produce amphotropic viral particles that are
able to infect a broad range of mammalian cells, including those ofhuman
origin. The potential biohazard due to the possible generation of replication
competent viruses has stimulated the design of new packaging celllines that
eliminate the recombination events leading to helper virus production.
With the ecotropic packaging celllines \j/CRE (15) and GP+E86 (16) helper
virus rescue has never been reported. These packaging celllines should preferentially be used, when the target cells are of rodent origin. For the generattion of amphotropic viruses, the packaging cellline \j/CRIP {15) is probably a safe choice. The protocols described here for the first generation
packaging cells \j/2 and\j/ AM arealso applicable, when the new second generation packaging celllines are used. It is preferable to transfect the \j/2 cells
first and then use the ecotropic viral stocks to infect the \j/AM cells for the
production of amphotropic viral stocks. The reasons are:
• Infected cells, but not transfected cells, all produce infectious virus. Thus,
the highest titer virus stocks are produced from infected cells.
• Transfeetion of proviral DNAs apparently enhances the rate of recombination which Ieads to helper virus rescue, so transfection of the packaging cells which produce amphotropic virus (\j/AM, \j/CRIP and PA317)
should be avoided.
• \j/2 cells have a lower rate of helper-virus rescue than the \j/AM cells.
Therefore transfection of \j/2 carries only a small risk of generation of
replication-competent (helper) virus.
Note: For the transfection of \j/2 cells we usually use the DMSO shock method, but other methods such as lipofection, electroporation or calcium phosphate coprecipitation may be used.