Bax Bak DKO SV40 MEF (ATCC® CRL-2913)

Organism: Mus musculus  /  Cell Type: fibroblast  /  Tissue: embryonic fibroblast  / 

Organism Mus musculus
Tissue embryonic fibroblast
Cell Type fibroblast
Product Format frozen
Morphology fibroblast-like
Culture Properties adherent
Biosafety Level 2   [Cells contain SV40 viral DNA sequences]
Applications This cell line is useful to study molecular mechanism of cell apoptosis, BCL-2 family pathway, BAX and BAK function, as well as cells that are resistant to multiple apoptotic stimuli.
Storage Conditions liquid nitrogen vapor phase
Images CRL-2913 Micrograph
Derivation Cells are immortalized MEFs which generated from BAX and BAK double knockout mice.
Tumorigenic no
Comments

Cells are immortalized MEFs which generated from BAX and BAK double knockout mice (genotype: bax-/-, bak-/-)

BAX and BAK are pro-apoptotic BCL-2 family members. Cells are completely resistant to tBID-induced cytochrome c release and apoptosis.

Cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. 

Cells are resistant to Bim and Bad expression induced apoptosis. 

Soft agar focus-formation assay revealed oncoprotein Ras and the adenovirus-5 E1A induced focus formation.

This cell line is useful to study molecular mechanism of cell apoptosis, BCL-2 family pathway, BAX and BAK function, as well as cells that are resistant to multiple apoptotic stimuli. 

Complete Growth Medium The base medium for this cell line is ATCC-formulated IMDM Catalog No. 30-2005. To make the complete growth medium, add the following components to the base medium: 10 % Fetal Bovine Serum and 1x Non-essential amino acids.
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 1.0 to 2.0 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 4 X 103 to 1 X 104 viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C. At subculture, cell concentration is between 1 X 105 to 2 X 105 cells/cm2.

Subcultivation ratio: A subcultivation ratio of 1:8 to 1:30 is recommended.
Medium renewal: Every 2 to 3 days
Culture Conditions Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Name of Depositor Standley Korsmeyer
References

Wei M, et al. Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death. Science 292(5517): 727-30, 2001 PubMed: 11326099

Lindsten T, et al. The Combined Functions of Proapoptotic Bcl-2 Family Members Bak and Bax Are Essential for Normal Development of Multiple Tissues. Molecular Cell 6(6): 1389-1399, 2000 PubMed: 11163212

Zong WX, et al. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes Dev. 15(12): 1481-6, 2001 PubMed: 11410528

Cheng EH, et al. BCL-2, BCL-XL Sequester BH3 Domain-Only Molecules Preventing BAX- and BAK-Mediated Mitochondrial Apoptosis. Molecular Cell 8(3) : 705–711, 2001 PubMed: 11583631

Basic Documentation
Other Documentation
References

Wei M, et al. Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death. Science 292(5517): 727-30, 2001 PubMed: 11326099

Lindsten T, et al. The Combined Functions of Proapoptotic Bcl-2 Family Members Bak and Bax Are Essential for Normal Development of Multiple Tissues. Molecular Cell 6(6): 1389-1399, 2000 PubMed: 11163212

Zong WX, et al. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes Dev. 15(12): 1481-6, 2001 PubMed: 11410528

Cheng EH, et al. BCL-2, BCL-XL Sequester BH3 Domain-Only Molecules Preventing BAX- and BAK-Mediated Mitochondrial Apoptosis. Molecular Cell 8(3) : 705–711, 2001 PubMed: 11583631