PA317 (ATCC® CRL-9078)

Organism: Mus musculus, mouse  /  Tissue: embryo  / 

Permits and Restrictions

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Organism Mus musculus, mouse
Tissue embryo
Product Format frozen
Morphology fibroblast
Culture Properties adherent
Biosafety Level 1
Age embryo
Strain NIH/Swiss
Applications
Virions produced by this line have been used successfully to transfer genes into humans.
Packaging line for retroviruses.
Storage Conditions liquid nitrogen vapor temperature
Derivation

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.


Comments

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.

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.

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.

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%.
Subculturing Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
  6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:10
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.


Cryopreservation
Complete growth medium 95%; DMSO, 5%. Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Culture Conditions
Temperature: 37°C
Name of Depositor Fred Hutchinson Cancer Res. Cntr.
References

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

Miller AD. DNA contructs for retrovirus packaging cell lines. US Patent 4,861,719 dated Aug 29 1989

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

Giguere V, et al. Identification of a new class of steroid hormone receptors. Nature 331: 91-94, 1988. PubMed: 3267207

Scaglioni PP, et al. Posttranscriptional regulation of hepatitis B virus replication by the precore protein. J. Virol. 71: 345-353, 1997. PubMed: 8985356

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

Wang Y, et al. Leptin receptor action in hepatic cells. J. Biol. Chem. 272: 16216-16223, 1997. PubMed: 9195922

Lu Y, et al. Inhibition of HIV-1 replication using a mutated tRNA Lys-3 primer. J. Biol. Chem. 272: 14523-14531, 1997. PubMed: 9169409

Morgan SE, et al. Fragments of ATM which have dominant-negative or complementing activity. Mol. Cell. Biol. 17: 2020-2029, 1997. PubMed: 9121450

Clausen PA, et al. ETS-1 induces increased expression of erythroid markers in the pluripotent erythroleukemic cell lines K562 and HEL. Leukemia 11: 1224-1233, 1997. PubMed: 9264374

Fan H, et al. The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments. Proc. Natl. Acad. Sci. USA 94: 13181-13186, 1997. PubMed: 9371820

Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online at http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
References

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

Miller AD. DNA contructs for retrovirus packaging cell lines. US Patent 4,861,719 dated Aug 29 1989

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

Giguere V, et al. Identification of a new class of steroid hormone receptors. Nature 331: 91-94, 1988. PubMed: 3267207

Scaglioni PP, et al. Posttranscriptional regulation of hepatitis B virus replication by the precore protein. J. Virol. 71: 345-353, 1997. PubMed: 8985356

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

Wang Y, et al. Leptin receptor action in hepatic cells. J. Biol. Chem. 272: 16216-16223, 1997. PubMed: 9195922

Lu Y, et al. Inhibition of HIV-1 replication using a mutated tRNA Lys-3 primer. J. Biol. Chem. 272: 14523-14531, 1997. PubMed: 9169409

Morgan SE, et al. Fragments of ATM which have dominant-negative or complementing activity. Mol. Cell. Biol. 17: 2020-2029, 1997. PubMed: 9121450

Clausen PA, et al. ETS-1 induces increased expression of erythroid markers in the pluripotent erythroleukemic cell lines K562 and HEL. Leukemia 11: 1224-1233, 1997. PubMed: 9264374

Fan H, et al. The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments. Proc. Natl. Acad. Sci. USA 94: 13181-13186, 1997. PubMed: 9371820

Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online at http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm