TRF2 F/- Rosa26-CreERT2 (ATCC® CRL-3317)

Organism: Mus musculus, mouse  /  Tissue: E13.5 mouse embryo  / 

Permits and Restrictions

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Organism Mus musculus, mouse
Tissue E13.5 mouse embryo
Product Format frozen 1.0 mL
Morphology fibroblast-like
Culture Properties adherent
Biosafety Level 2 [Cells contain retroviral and SV40 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.

Strain B6/129
Applications DNA damage repair mechanisms, telomere biology
Storage Conditions vapor phase liquid nitrogen
Images Cell Micrograph of TRF2 F/-Rosa 26-CreERT2 cells, ATCC CRL-3317
Comments

This mouse embryonic fibroblast cell line possess loxP sites on either side of the first and second coding exon of mouse TRF2 (Terf2) gene. This MEF line also expresses Cre-ERT2 from one Rosa26 locus. TRF2 knockout can be done by Cre recombinase transduction or 0.5 uM 4-hydroxytamoxifen treatment for 6hr of the MEFs. This inducible TRF2 knockout MEF line is useful in studies of telomere biology.

Development of the system:
A targeting vector containing neomycin resistance and herpes simplex virus thymidine kinase genes under the control of the phosphoglyceryl kinase gene promoter was utilized in the construction of this mutant. A loxP site was inserted upstream of exon 1. Another loxP site was inserted downstream of exon 2. This construct was electroporated into 129P2/OlaHsd derived E14 embryonic stem (ES) cells which were transiently transfected with a Cre recombinase vector to remove the selection cassette. Correctly targeted ES cells were injected into recipient blastocysts.

Complete Growth Medium

The base medium for this cell line is DMEM (ATCC® 30-2002™). To make the complete medium add the following components to 500 mL of the base medium:

  • 56 mL fetal bovine serum (FBS; ATCC® 30-2020™) for a final concentration of 10%
  • 5.6 mL L-glutamine (200 mM; ATCC® 30-2214™) for a final concentration of 2 mM
  • 5.6 mL NEAA (Nonessential Amino Acids, 100X; Gibco cat# 11140050) for a final concentration of 0.1 mM
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. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that 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.
    Cultures can be established between 2 x 104 and 4 x 104 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 2 X 104 and 2.4 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 1 x 106 cells
Volume 1.0 mL
Sterility Tests no growth
Viability ≥ 50%
Name of Depositor T De Lange
Year of Origin 2015
References

Dimitrova N, et al. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature 456(7221): 524-528, 2008. PubMed: 18931659

Celli G, de Lange T. DNA processing not required for ATM activation or the telomere damage response after conditional deletion of mouse TRF2. Nat Cell Biol 7(7): 712-718, 2005. PubMed: 15968270

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • For-profit customers must obtain a research use license agreement from the Contributor prior to shipment.
  • 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
Other Documentation
Restrictions

This material’s use is governed by the MTA and the following restrictions: (1) Prior to purchase, for-profit entities must obtain a research use license from Rockefeller University and (2) when a Commercial Use is contemplated, for-profit and non-profit entities must obtain a Commercial Use license from Rockefeller University. Rockefeller University will be informed of all customers that purchase this product. For information on executing a license to use this product for research (for-profit entities) or Commercial Use (for-profit and non-profit entities), contact Rockefeller University, Office of Technology Transfer, 1230 York Avenue, New York, NY 10065 Attn: Nidhi Sabharwal, Assistant Director, Marketing & Licensing at nsabharwal@rockefeller.edu.

References

Dimitrova N, et al. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature 456(7221): 524-528, 2008. PubMed: 18931659

Celli G, de Lange T. DNA processing not required for ATM activation or the telomere damage response after conditional deletion of mouse TRF2. Nat Cell Biol 7(7): 712-718, 2005. PubMed: 15968270