| Comments |
The genotype of this cell line has recently been determined to be DNA polymerase beta mutant (null) and DNA polymerase iota mutant (null).
The pol-beta deficient fibroblasts were immortalized by transfection with the DNA plasmid construct ptsA58H which contains coding sequences for both a hygromycin-resistance gene and tsA58H, a temperature-sensitive SV40 T antigen.
Cells were selected in the presence of 0.080 mg/ml hygromycin for 21 days and further selected by limiting dilution.
The cell line is deficient in base excision repair due to homozygous deletion of the promoter and exon I in the pol-bet a gene.
The deletion of pol-beta is mediated by the Cre-loxP recombination.
This deletion event is reversible and therefore this cell line is ideal for site-specific recombination using the Cre-lox system.
The contributor of this line has demonstrated that insertion of a single-copy of a variety of cDNAs can be site-specifically reinserted at the pol-beta locus, and that this is a stable event. |
| 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 34°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 34°C.
Subcultivation Ratio: 1:10 to 1:15
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 |
Gu H, et al. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265: 103-106, 1994. PubMed: 8016642
Sobol RW, et al. Requirement of mammalian DNA polymerase-beta in base-excision repair. Nature 379: 183-186, 1996. PubMed: 8538772
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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