NIH/Swiss

The PA317 cell line was derived from TK- NIH/3T3 cells by cotransfection with packaging construct DNA (pPAM3) carried in pBR322 and the herpes simplex virus thymidine kinase (TK) gene carried in pBR322.
Introduction of retroviral vectors into these cells, by infection or by transfection, results in production of retrovirus virions with an amphotropic host range that are capable of infecting cells of many mammalian species.
Virions produced by this line have been used successfully to transfer genes into humans.
The percentage of PA317 cells that are capable of packaging retroviral vectors decreases slowly with continued passaging of the cell line, presumably due to the loss of the transfected DNA used to create the line.
Brief selection (about 5 days) in medium containing 0.03 mM hypoxanthine, 0.001 mM amethopterin (methotrexate), and 0.02 mM thymidine will select for cells that retain the packaging function.
After selection, the cells should be grown in HT medium (0.03 mM hypoxanthine, 0.02 mM thymidine) for 4 days to dilute any residual amethopterin.
Cells shipped from the ATCC have been selected in medium containing 0.03 mM hypoxanthine, 0.001 mM amethopterin (methotrexate), and 0.02 mM thymidine prior to freezing, and should be grown in HT medium for 4 days after receipt.
After this, the cells are stable for at least 1 month in the absence of further selection.

Protocol: Remove medium, and rinse with 0.25% trypsin, 0.03% EDTA solution. Remove the solution and add an additional 1 to 2 ml of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 37C) until the cells detach. Add fresh culture medium, aspirate and dispense into new culture flasks.

Subcultivation ratio: A subcultivation ratio of 1:10 is recommended

Medium renewal: 2 to 3 times per week

Recommended medium (without the additional supplements or serum described under ATCC Medium): ATCC 30-2002
recommended serum: ATCC 30-2020