| Derivation |
This line was derived from the ATCC PTA-803 cell line that was derived from HeLa (ATCC CCL-2) [U.S. Pat. 6,365,394]. The line was stably transfected with plasmid constructs carrying a 3.4 kb DNA fragment of Ad 5 genome spanning 511 to 3924 bp (E1a and E1b open reading frames and part of the pIX gene) under the control of a phosphoglycerate kinase (PGK) promotor. The cells contain multiple copies of the plasmids, which carry multiple copies of E1a and E1b genes. The plasmid also contains the neomycin resistance gene. |
| Comments |
This line was derived from the ATCC PTA-803 cell line that was derived from HeLa (ATCC CCL-2) [U.S. Pat. 6,365,394]. The line was stably transfected with plasmid constructs carrying a 3.4 kb DNA fragment of Ad 5 genome spanning 511 to 3924 bp (E1a and E1b open reading frames and part of the pIX gene) under the control of a phosphoglycerate kinase (PGK) promotor. The cells contain multiple copies of the plasmids, which carry multiple copies of E1a and E1b genes. The plasmid also contains the neomycin resistance gene. |
| 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 |
Gao GP, et al. A cell line for high-yield production of E1-deleted adenovirus vectors without the emergence of replication-competent virus. Hum. Gene Ther. 11: 213-219, 2000. PubMed: 10646652
Gao G, Wilson JM. Cell lines and constructs useful in production of E1-deleted adenoviruses in absence of replication competent adenovirus. US Patent 6,365,394 dated Apr 2 2002
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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