MB19tsA, clone 2B2 [Mbeta19tsA, clone 2B2] (ATCC® CRL-2308)

Organism: Mus musculus, mouse  /  Cell Type: fibroblast SV40 large T antigen transfected  /  Tissue: embryo  / 

Permits and Restrictions

View Permits

Organism Mus musculus, mouse
Tissue embryo
Cell Type fibroblast SV40 large T antigen transfected
Product Format frozen
Morphology fibroblast
Culture Properties adherent
Biosafety Level 2  [Cells contain SV-40 viral sequences]

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Age 10.5 days gestation
Applications
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.
This deletion event is reversible and therefore this cell line is ideal for site-specific recombination using the Cre-lox system.
Storage Conditions liquid nitrogen vapor phase
Derivation
MB19tsA, clone 2B2 is a fibroblast-like cell line derived from primary cultures of DNA polymerase beta (pol-beta) deficient fibroblasts isolated from embryos from heterozygous females ( beta-polymerase +/-) at day 10.5 of gestation.
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.
Complete Growth Medium Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 0.08 mg/ml hygromycin B, 90%; fetal bovine serum, 10%
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.
  1. Remove and discard culture medium.
  2. 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.
  3. 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.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
  6. 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.

Cryopreservation

Complete growth medium described above supplemented with 5% (v/v) DMSO.  Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Culture Conditions
Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO2), 5%
Name of Depositor RW Sobol, SH Wilson
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.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
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.