POT1a F/F POT1b F/F(#20431-5) (ATCC® CRL-3323)

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
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 liquid nitrogen vapor phase
Images Cell Micrograph of POT1a F/F POT1b F/F(#20431-5), ATCC CRL-3323
Comments

These MEFs possess loxP sites on either side of the third coding exon of the Pot1a and Pot1b genes. Pot1a and POT1b genes can be deleted by transduction of Cre recombinase to the MEFs. This inducible POT1a POT1b knockout MEF line is useful in studies of telomere biology.

Immortalization process: Infection of pBabe neo SV40-LT.

The MEFs were harvested at E13.5 and were infected with pBabe Neo SV40-LT for immortalization. Cells were cryopreserved at PD 30. One of the vials was defrosted, expanded to two 15 cm dishes, and 10 vials of frozen stock were made for this deposition. One vial was defrosted and tested for recovery rate, mycoplasma contamination, and genotype.

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:

  • 57 mL fetal bovine serum (FBS; ATCC 30-2020) for a final concentration of 10%
  • 5.7 mL L-glutamine (200 mM; ATCC 30-2214) for a final concentration of 2 mM
  • 5.7 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 1x 104 and 5 x 104 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 1 X 104 and 2 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 1.0 to 2.0 x 106 cells
Volume 1.0 mL
Sterility Tests Bacteria and Yeast: No growth
Mycoplasma: No growth
Viral Testing Hepatitis B: None detected
Cytomegalovirus: None detected
Human Immunodeficiency virus: None detected
EBV: None detected
HPV: None detected
Viability ≥ 50%
Name of Depositor T De Lange
Year of Origin 2009
References

Kibe T, et al. Telomere Protection by TPP1 is mediated by POT1a and POT1b. Mol Cell Biol 30(4): 1059-1066, 2010. PubMed: 19995905

Hockemeyer D, et al. Recent expansion of the telomeric complex in rodents: Two distinct POT1 proteins protect mouse telomeres. Cell 126(1): 63-77, 2006. PubMed: 16839877

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

Kibe T, et al. Telomere Protection by TPP1 is mediated by POT1a and POT1b. Mol Cell Biol 30(4): 1059-1066, 2010. PubMed: 19995905

Hockemeyer D, et al. Recent expansion of the telomeric complex in rodents: Two distinct POT1 proteins protect mouse telomeres. Cell 126(1): 63-77, 2006. PubMed: 16839877