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2A (ATCC® CRL-12013)

Organism: Homo sapiens, human  /  Tissue: kidney  / 

Permits and Restrictions

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Organism Homo sapiens, human
Tissue kidney
Product Format frozen
Culture Properties adherent
Biosafety Level 2 Cells contain adenoviral DNA sequences

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Age fetus
Applications
The 2A amphotropic retroviral packaging cell line was derived from the human embryonic kidney line, 293 (see ATCC CRL-1573).
After selection for transfected cells, individual drug resistant cell colonies were expanded, analyzed and selected for overexpression of MoMLV gag/pol.
Storage Conditions liqud nitrogen vapor phase
Disclosure This material is cited in a US or other Patent and may not be used to infringe the claims. Depending on the wishes of the Depositor, ATCC may be required to inform the Patent Depositor of the party to which the material was furnished. This material may not have been produced or characterized by ATCC.
Derivation
The 2A amphotropic retroviral packaging cell line was derived from the human embryonic kidney line, 293 (see ATCC CRL-1573).
Comments
The 2A amphotropic retroviral packaging cell line was derived from the human embryonic kidney line, 293 (see ATCC CRL-1573).
293 cells were co-transfected with the methotrexate resistance vector, pFR400 and the MoMLV gag/pol expression vector, pSCV10.
After selection for transfected cells, individual drug resistant cell colonies were expanded, analyzed and selected for overexpression of MoMLV gag/pol.
293 (clone 2-3) was co-transfected with the phleomycin resistance vector, pUT507 and the amphotropic envelope expression vector, pMLPenvAmSph and subsequently cloned.
The 2A clone was selected for expression of relatively high levels of both gag/pol and amphotropic envelope proteins.
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:
  • O.1 mM Non-Essential Amino Acids (NEAA)
  • 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: 1:4 to 1:8
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 described above supplemented with 5% (v/v) DMSO.  Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Culture Conditions
Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO2), 5%
Name of Depositor Chiron Viagene, Inc.
Deposited As human
U.S. Patent Number
Disclosure This material is cited in a US or other Patent and may not be used to infringe the claims. Depending on the wishes of the Depositor, ATCC may be required to inform the Patent Depositor of the party to which the material was furnished. This material may not have been produced or characterized by ATCC.
References

DePolo NJ, et al. The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent. J. Virol. 73: 6708-6714, 1999. PubMed: 10400768

Barber JR, et al. Retroviral packaging cell lines. US Patent 5,591,624 dated Jan 7 1997

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

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.

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

DePolo NJ, et al. The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent. J. Virol. 73: 6708-6714, 1999. PubMed: 10400768

Barber JR, et al. Retroviral packaging cell lines. US Patent 5,591,624 dated Jan 7 1997

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

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.