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Cell Biology
ATCC® Number: CRL-9078™    Price: $329.00
Designations: PA317
Depositors:  Fred Hutchinson Cancer Res. Cntr.
Biosafety Level: 1
Shipped: frozen
Medium & Serum: See Propagation
Growth Properties: adherent
Organism: Mus musculus (mouse)
Morphology: fibroblast
Source: Organ: embryo
Strain: NIH/Swiss
Permits/Forms: In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.
Applications: transfection host (Roche FuGENE® Transfection Reagents)
Packaging line for retroviruses.
Age: embryo
Comments: Derived from TK- NIH/3T3 cells by cotransfection with packaging construct DNA, pPAM3, carried in pBR322 and herpes simplex virus thymidine kinase (TK) gene carried in pBR322.
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.
Propagation: ATCC complete growth medium: The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Temperature: 37.0°C
Subculturing: 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
Preservation: Freeze medium: Complete growth medium 95%; DMSO, 5%
Storage temperature: liquid nitrogen vapor temperature
Related Products: Recommended medium (without the additional supplements or serum described under ATCC Medium):ATCC 30-2002
recommended serum:ATCC 30-2020
References: 1608: Miller AD, Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6: 2895-2902, 1986. PubMed: 3785217
21933: Miller AD. DNA contructs for retrovirus packaging cell lines. US Patent 4,861,719 dated Aug 29 1989
22890: Rosenberg SA, et al. Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 323: 570-578, 1990. PubMed: 2381442
23795: Giguere V, et al. Identification of a new class of steroid hormone receptors. Nature 331: 91-94, 1988. PubMed: 3267207
27269: Scaglioni PP, et al. Posttranscriptional regulation of hepatitis B virus replication by the precore protein. J. Virol. 71: 345-353, 1997. PubMed: 8985356
33067: Lai CF, et al. Receptors for interleukin (IL)-10 and IL-6-type cytokines use similar signaling mechanisms for inducing transcription through IL-6 response elements. J. Biol. Chem. 271: 13968-13975, 1996. PubMed: 8662928
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