PERK-KO-DR (ATCC® CRL-2976)

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

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Organism Mus musculus, mouse
Cell Type fibroblast
Product Format frozen
Morphology fibroblast-like
Culture Properties adherent
Biosafety Level 2
Age embryo, 13.5 days gestation
Applications
ER stress; unfolded protein response signaling pathway
Storage Conditions liquid nitrogen vapor phase
Images
Derivation
The mouse embryonic fibroblast (MEF) cell line, PERK-KO-DR (CRL-2976), was established from a 13.5 day-old PERK -/- mouse embryo by SV-40 immortalization. These cells lack the Endoplasmic Reticulum (ER) localized stress induced kinase, PERK, which mediates signaling from the stressed ER to the translational apparatus by phosphorylating eukaryotic initiation factor 2a (eIF2a). Phosphorylation of eIF2a results in a global inhibition of protein synthesis. PERK is a major regulator of the unfolded protein response (UPR), and as such, these PERK-KO-DR cells are very useful to investigate ER stress and UPR signaling in a variety of contexts. DR-Wildtype cells, ATCC CRL-2977, are available for use as a control. In addition, GCN2 knockout MEF cell line ATCC CRL-2978 and CHOP knockout MEF cell line ATCC CRL-2979 are two related important tools for ER stress and UPR studies.
Genes Expressed
protein kinase RNA-like endoplasmic reticulum kinase (PERK), not expressed
Comments
The mouse embryonic fibroblast (MEF) cell line, PERK-KO-DR (CRL-2976), was established from a 13.5 day-old PERK -/- mouse embryo by SV-40 immortalization. These cells lack the Endoplasmic Reticulum (ER) localized stress induced kinase, PERK, which mediates signaling from the stressed ER to the translational apparatus by phosphorylating eukaryotic initiation factor 2a (eIF2a). Phosphorylation of eIF2a results in a global inhibition of protein synthesis. PERK is a major regulator of the unfolded protein response (UPR), and as such, these PERK-KO-DR cells are very useful to investigate ER stress and UPR signaling in a variety of contexts. DR-Wildtype cells, ATCC CRL-2977, are available for use as a control. In addition, GCN2 knockout MEF cell line ATCC CRL-2978 and CHOP knockout MEF cell line ATCC CRL-2979 are two related important tools for ER stress and UPR studies.
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
Protocol: 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 37.0°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 37.0°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: 37.0°C
Atmosphere: air, 95%; carbon dioxide (CO,2), 5%
Name of Depositor D Ron and H Harding
Year of Origin 2000
References

Harding HP, et al. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397(6716): 271-274, 1999. PubMed 9930704

Harding HP, et al. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5(5): 897-904, 2000. PubMed: 10882126

Harding HP, et al. Diabetes mellitus and exocrine pancreatic dysfunction in perk -/- mice reveals a role for translational control in secretory cell survival. Mol. Cell 7(6): 1153-1163, 2001. PubMed: 11430819

Kimball SR, et al. Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. Am. J. Physiol. Cell Physiol. 284(2): C273-C284, 2003. PubMed: 12388085

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
Other Documentation
References

Harding HP, et al. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397(6716): 271-274, 1999. PubMed 9930704

Harding HP, et al. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5(5): 897-904, 2000. PubMed: 10882126

Harding HP, et al. Diabetes mellitus and exocrine pancreatic dysfunction in perk -/- mice reveals a role for translational control in secretory cell survival. Mol. Cell 7(6): 1153-1163, 2001. PubMed: 11430819

Kimball SR, et al. Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. Am. J. Physiol. Cell Physiol. 284(2): C273-C284, 2003. PubMed: 12388085