56B3 [derivative of CRL-11117] (CRL-2542)

Organism: Mus musculus, mouse  /  Cell Type: embryonic stem cell  / 

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Organism Mus musculus, mouse
Cell Type embryonic stem cell
Product Format frozen
Culture Properties adherent
Biosafety Level 1
Age embryo
Strain 129/Sv+c/+p
Applications
These cell lines were used to produce mutant mice with germ line transmission for lck disruption.
Storage Conditions liquid nitrogen vapor phase
Comments
The replacement-type vector (PmlckBSNeo2.3) was introduced into D3 embryonic stem (ES) cells by electroporation to disrupt the lck locus.
Two cell lines, 59B5 (ATCC-CRL-11115) and 56B3 (ATCC-CRL-11117), were generated that were deficient for the lck gene.
These cell lines were used to produce mutant mice with germ line transmission for lck disruption.
Heterozygous mice were inbred to obtain mice homozygous for the disrupted lck gene.
A culture submitted to the ATCC as CRL-11117 in September of 1992 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with BM Cycline.
The cured cell line is available as CRL-2542. The original patent deposit is available as CRL-11117.
The line should be grown on feeder layers of mitomycin C treated (0.01 mg/ml for 90 minutes) primary mouse embryonic fibroblasts or STO cells (see ATCC CRL-1503 or ATCC 56-X.2, MITC-STO cells).
Complete Growth Medium Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose and supplemented with 0.1 mM 2-mercaptoethanol and 10mM HEPES, 85%; fetal bovine serum, 15%
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 37°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.
    Note: The line should be grown on feeder layers of irradiated (3000 rads) or mitomycin C treated (0.01 mg/mL for 90 minutes) primary mouse embryonic fibroblasts or STO cells (see ATCC CRL-1503 or ATCC 56-X, irradiated STO cells).
  6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:3 to 1:6
Medium Renewal: Three times a week 

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 TW Mak
U.S. Patent Number
References

Mak TW. Mouse having a disrupted lck gene. US Patent 5,625,122 dated Apr 29 1997

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 at http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm

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

Mak TW. Mouse having a disrupted lck gene. US Patent 5,625,122 dated Apr 29 1997

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 at http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm