PA317

Transfection and virus studies: herpes virus

Mouse fibroblast with herpes TK gene

The cell line PA317 was generated by co-transfecting NIH 3T3 TK- cells with retrovirus packaging construct DNA (pPAM3) and the herpes simplex virus thymidine kinase (TK) gene carried as a fragment in pBR322. The construct pPAM3 contains several mutations in addition to deletion of the packaging signal. These features appear to prevent the production of helper virus and the transfer of packaging function. Transfection or infection with retroviral vectors results in the production of retrovirus virions with an amphotropic host range. These are capable of infecting mammalian cells and have been applied to transfer genes into humans. PA317 cells will loose their packaging ability during long-term culture. Therefore, a selection should be included routinely and before cryopreservation of cells. The selection procedure includes growth in HAT-containing medium (0.03mM hypoxanthine, 0.001mM amethopterin, 0.02mM thymidine) for 5 days. To dilute residual amethopterin cells should then be cultured for 4 days in HT containing medium (0.03mM hypoxanthine, 0.02mM thymidine). Cells are stable for at least 1 month after selection.

DMEM + 2mM Glutamine + 10% Foetal Bovine Serum (FBS).

Split sub-confluent cultures (70-80%) 1:2 to 1:3 i.e. seeding at 3-5x10,000 cells/cm² using 0.25% trypsin; 5% CO₂; 37°C; Grow in HAT for 5 days followed by 4 days in HT once a month and prior to cryopreservation

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This cell line is part of the European Collection of Authenticated Cell Cultures (ECACC), an international repository managed by the United Kingdom Health Security Agency (UKHSA). No licensing agreement is required when either this cell line or the DNA extracted from it are used for internal research purposes only. Any other use of these products is prohibited without the express written permission of UKHSA. Inquiries regarding authorized use of this cell line or its genomic DNA may be directed to culturecollections@ukhsa.gov.uk.