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B-MEKDD 116; Mammary Epithelium; Mouse (Mus musculus) Item ID: ATCcrl3069

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Organism Mus musculus, mouse
Tissue breast epithelium (mammary gland)
Cell Type epithelial cell
Product Format frozen
Morphology epithelial-like
Culture Properties adherent
Biosafety Level 2

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.
Gender female
Applications This cell line stably expresses constitutively activated form of MEK1, and can be used in MEK-MAPK pathway studies. This cell line is tumorigenic, and can be used to build a breast cancer in vivo model.
Storage Conditions liquid nitrogen vapor phase
Derivation

B-MEKDD 116 was produced by transfecting parental EpH4, a mouse mammary epithelial cell line, with an expression vector containing Glu-Glu epitope-tagged phosphorylation site MEK1 mutant (MEKDD).  After selection in complete medium supplemented with 1 mg/mL G418 for 14 days, individual clones were isolated and expanded.
Oncogene Constitutively activated form of MEK1, expressed
Genes Expressed Glu-Glu tagged phosphorylation site MEK1 mutant (MEKDD), verified at ATCC
Cellular Products stably expresses Glu-Glu tagged MEKDD, which is constitutively activated form of MEK1 (Asp218/Asp222 MEK1 phosphorylation site mutant)
Tumorigenic Yes
Comments

B-MEKDD 116 was produced by transfecting parental EpH4, a mouse mammary epithelial cell line, with an expression vector containing Glu-Glu epitope-tagged phosphorylation site MEK1 mutant (MEKDD).  After selection in complete medium supplemented with 1 mg/mL G418 for 14 days, individual clones were isolated and expanded.

Activation of MEK1 is mediated through phosphorylation of Ser218 and Ser222 by members of the Raf family of kinases. 

This cell line stably expresses constitutively activated form of MEK1 (Asp218/Asp222 MEK1 phosphorylation site mutant), and can be used in MEK-MAPK pathway studies. This cell line is tumorigenic, and can be used to build a breast cancer in vivo model. There are four additional related cell lines: EpH 1424 cell line (ATCC CRL-3071), EpH4 1424.1 cell line (ATCC CRL-3209), EpH4 1424.2 cell line (ATCC CRL-3210), and EpH4-Ev cell line (ATCC CRL-3063).

The B-MEKDD 116 cell line produces the constitutively activated MEK1 mutant MEKDD which has been tagged with Glu-Glu, verified at ATCC.
Complete Growth Medium The base medium for this cell line is ATCC-formulated DMEM Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: 10% Bovine Calf Serum and 200 mcg/mL G418
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 8 x 103 to 1.5 x 104 viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C. Subculture when the cell concentration is between 8 x 104 to 1.5 x 105 cells/cm2.
Subcultivation ratio: A subcultivation ratio of 1:6 to 1:12 is recommended.Medium renewal: Every 2 to 3 days
Cryopreservation Freeze medium: complete growth medium supplemented with an additional 10% bovine calf serum and 10% DMSOStorage temperature: liquid nitrogen vapor phase
Culture Conditions Temperature: 37°CAtmosphere: air, 95%; carbon dioxide (CO2), 5%
Name of Depositor P Leder
References

Pinkas J, et al. MEK1 signaling mediates transformation and metastasis of EpH4 mammary epithelial cells independent of an epithelial to mesenchymal transition. Cancer Res. 62(16): 4781-90. PubMed: 12183438

Pinkas J, et al. Bcl-2-mediated cell survival promotes metastasis of EpH4 betaMEKDD mammary epithelial cells. Mol. Cancer Res. 2(10): 551-6, 2004. PubMed: 15498929

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