MEF (DR4) (ATCC® SCRC-1045)

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
Culture Properties Adherent
Biosafety Level 1
Age 14 days gestation embryo
Gender Male and female mixed
Strain DR4
Applications

Can be used to produce feeder cells with multiple drug resistance.

SCRC-1045 cells can be used as a feeder layer to support the growth of engineered embryonic stem (ES) cells with multiple drug selections and for the maintenance of ES cells in the undifferentiated state. 


Storage Conditions liquid nitrogen vapor phase
Derivation The cell line was established by ATCC in 2003 from embryonic day 14 (E14) DR4 mouse embryos obtained from The Jackson Laboratory (Stock# 003208).  Mouse embryo fibroblasts (MEFs) prepared from the DR4 mouse strain are resistant to neomycin, hygromycin, puromycin, and 6-thioguanine. 

Dr. Rudolf Jaenisch at the Massachusetts Institute of Technology developed the DR4 mouse strain by the intercrossing of three different strains: one bearing resistance genes neoR and puroR, a second bearing the resistance gene hygR, and a third bearing a natural deletion encompassing the Hprt gene. A series of matings incorporated all 4 drug resistance genes into the strain (Tucker et al., 1997; PubMed: 9278500). The background of the original DR4 strain was a mix of 129S4/SvJae, 129P2/OlaHsd, BALB/c, and C57BL/6.

Clinical Data
male and female mixed
Comments
The growth of these cells should be arrested before being used as a feeder layer. ATCC has successfully irradiated and treated the cells with Mitomycin C for use as a feeder layer. If the MEFs are being used as a feeder layer for ES cells, it is not recommended to use them past passage no. 7 (P7).
ATCC tested that this cell line is resistant to:
  • G 418 (neomycin): 200 microgm/mL
  • Puromycin: 0.4 microgm/mL
  • Hygromycin: 110 microgm/mL
  • 6-Thioguanine: 2.5 microgm/mL
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:
  • fetal bovine serum to a final concentration of 15%

  • This medium is formulated for use with a 5% CO2 in air atmosphere. (Standard DMEM formulations contain 3.7 g/L sodium bicarbonate and a 10% CO2 in air atmosphere is then recommended).
    Subculturing To insure the highest level of viability, be sure to warm media and Trypsin / EDTA to 37ºC before using it on the cells. Cells should be split when they reach confluency. A split based on seeding density of 6 X 103 cells/cm2 is recommended.

     Note: Volumes used in this protocol are for 75cm2 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 5.0 mL 1XPBS (ATCC Catalog No. SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
    3. Add 3.0 mL 0.25% Trypsin-0.53 mM EDTA solution (ATCC Catalog No. 30-2101) solution to the flask and incubate for 2 minutes. Gently tap the flask and observe cells under an inverted microscope. Cells usually detach in 1 to 2 minutes.
    4. Add 3.0 mL complete growth medium and rinse the surface of the flask to detach all the cells. Gently pipette up and down will break cell clumps.
    5. Transfer all cell suspension into a centrifuge tube and centrifuge at 270 xg for 5 minutes.
    6. Remove and discard the supernatant.
    7. Add complete growth medium to the cell pellet and with 10 mL pipette gently resuspend the cells gently to create a single-cell suspension.
    8. Adjust volume as needed to seed vessels at approximately 6 X103 cells/cm2.
    9. Place flasks in incubator at 37° with 5% CO2 in air atmosphere.

    Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.  

    Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:7 is recommended

    Medium Renewal: Twice a week or when pH decreases
    Cryopreservation
    Complete growth medium supplemented with an additional 40% FBS and 10% (v/v) DMSO
    Cell culture tested DMSO is available as ATCC Catalog No. 4-X.
    Culture Conditions
    Atmosphere: Air, 95%; carbon dioxide (CO2), 5%
    Temperature: 37°C
    Name of Depositor ATCC
    Year of Origin 2003
    References

    Tucker KL, et al. A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res. 25: 3745-3746, 1997. PubMed: 9278500

    Nagy A, et al. Manipulating The Mouse Embryo: A Laboratory Manual. Third Edition: Cold Spring Harbor Press; 2003.

    Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

    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
    Restrictions

    Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact ATCC's Office of Licensing and Business Development at licensing@atcc.org or 703-365-2773.

    References

    Tucker KL, et al. A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res. 25: 3745-3746, 1997. PubMed: 9278500

    Nagy A, et al. Manipulating The Mouse Embryo: A Laboratory Manual. Third Edition: Cold Spring Harbor Press; 2003.

    Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.