Comments |
The RKO-AS45-1 cell line was generated from the colon carcinoma RKO (ATCC CRL-2577) cell line by transfection with the open reading frame portions of human cDNAs for GADD45 cloned into the expression vector pCMV. The cells contain a stably integrated cytomegalovirus (CMV) promotor driven expression vector. The cells overexpress Gadd45 RNA and protein. The GADD45 gene is p53-regulated and the Gadd45 protein interacts with proteins involved in both cell-cycle regulation and DNA repair. The RKO-AS45-1 cell line has reduced ability to repair U.V.-induced DNA damage. The RKO-AS45-1 cell line can be used together with its parental cell line, RKO (ATCC CRL-2577) to investigate the effects of Gadd45 on DNA repair, apoptosis and drug sensitivity. |
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: 1:10
Medium Renewal: Every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005. |
References |
Smith ML, et al. Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin. Oncogene 13: 2255-2263, 1996. PubMed: 8950993
Smith ML, et al. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 266: 1376-1380, 1994. PubMed: 7973727
Bae I, et al. An abnormality in the p53 pathway following gamma-irradiation in many wild-type p53 human melanoma lines. Cancer Res. 56: 840-847, 1996. PubMed: 8631022
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.
Smith ML, et al. Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin. Oncogene 13: 2255-2263, 1996. PubMed: 8950993
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