| 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. |
| 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. |
| 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.
- Remove and discard culture medium.
- 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.
- 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.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels.
- 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. |
| 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.
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