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.

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.

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.

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 Dulbeccos 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.

Freeze medium: fetal bovine serum, 90%; DMSO, 10%

Storage temperature: liquid nitrogen vapor phase

Temperature: 37°C

Atmosphere: air, 95%; carbon dioxide (CO₂), 5%