GCN2-KO-DR (ATCC® CRL-2978)

Organism: Mus musculus, mouse  /  Cell Type: fibroblast  /  Tissue: embryo  / 

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Organism Mus musculus, mouse
Tissue embryo
Cell Type fibroblast
Product Format frozen
Morphology fibroblast-like
Culture Properties adherent
Biosafety Level 2
Age embryo, 13.5 days gestation
Applications
GCN2 is a major regulator of the endoplasmic reticulum unfolded protein response (UPR) and as such, these KO cells are a useful tool for studying UPR. DR-Wildtype cells, ATCC CRL-2977, are available for use as a control.
The cells lack the nutritional stress-linked kinase GCN2, which phosphorylates eIF2a (alpha subunit of eukaryotic initiation factor 2) and complements PERK. GCN2-/- cells have been used to assess the role of eIF2a phosphorylation in a variety of pathophysiological conditions.
Storage Conditions liquid nitrogen vapor phase
Derivation
The mouse embryonic fibroblast (MEF) cell line, CRL-2978 (GCN2-KO-DR), was established from a 13.5 day-old GCN2-/- mouse embryo by SV-40 immortalization.
Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium:
  • O.1 mM Non-Essential Amino Acids (NEAA)
  • 0.05mM 2-Mercaptoethanol
  • fetal bovine serum to a final concentration of 10%

Subculturing
Volumes used in this protocol are for 75 cm2 flasks; 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 Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM 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. An inoculum of 1 X 103 to 2 X 103 viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C.
Subcultivation ratio: A subcultivation ratio of 1:10 to 1:30 twice weekly is recommended.
Medium renewal: Every 2 to 3 days.
Cryopreservation
Freeze medium: fetal bovine serum, 90%; DMSO, 10%
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Name of Depositor D Ron and H Harding
Year of Origin 2000
References

Harding HP, et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6(5): 1099-1108, 2000. PubMed: 11106749

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

Harding HP, et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6(5): 1099-1108, 2000. PubMed: 11106749