| Derivation |
This line is an amphotropic retrovirus packaging cell line derived from the human embryonic kidney line, 293 (ATCC CRL-1573). 293 cells were co-transfected with the pIK6.1MCVampac plasmid (ATCC 75483) encoding the gag and pol genes from ectropic MMLV and the envelope gene from the 4070A amphotropic MLV and MC1 neo [U.S. Pat. 5,858,740]. The line does not depend on continued G418 selection to maintain the packaging genome over time. |
| Comments |
This line is an amphotropic retrovirus packaging cell line derived from the human embryonic kidney line, 293 (ATCC CRL-1573). 293 cells were co-transfected with the pIK6.1MCVampac plasmid (ATCC 75483) encoding the gag and pol genes from ectropic MMLV and the envelope gene from the 4070A amphotropic MLV and MC1 neo [U.S. Pat. 5,858,740]. The line does not depend on continued G418 selection to maintain the packaging genome over time. |
| 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. An inoculum of 2 x 103 to 6 x 103 viable cells/cm2 is recommended. Maintain cultures at a cell concentration between 2 x 103 and 2.5 x 105 cells/cm2.
NOTE: Coat vessels with 0.1% pocine gelatin for 30 minutes to enhance attachment.
- Incubate cultures at 37°C.
Subcultivation Ratio: 1:4 to 1:6
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 |
Finer MH, et al. Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells. US Patent 5,858,740 dated Jan 12 1999
Finer MH, et al. Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells. US Patent 6,506,604 dated Jan 14 2003
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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