MEF (DR4)細胞
器官來源： 胚胎
生長狀態： 貼壁生長
年限： 14 days gestation embryo
ATCC Number： SCRC-1045?
運輸方式： 凍存運輸
數量： 大量
細胞類型： 成纖維細胞
是否是腫瘤細胞： 0
物種來源： 小鼠
細胞形態： 成纖維樣
Designations: MEF (DR4)
Depositors: ATCC
MEF (DR4)細胞Biosafety Level: 1
Shipped: frozen
Medium & Serum: See Propagation
Growth Properties: adherent
Organism: Mus musculus
Morphology: fibroblast
Source: Organ: embryo
Cell Type: fibroblast
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Isolation: MEF (DR4)細胞Isolation date: 2003
Applications: can be used to produce feeder cells
Age: 14 days gestation embryo
Gender: male and female mixed
Comments: 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 are resistant to common concentrations of the drugs G418, 6-thioguanine, puromycin, and hygromycin. The DR4 strain of mice was developed by Rudolf Jaenisch at the Massachusetts Institute of Technology. The DR4 strain was prepared 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 [PubMed: 9278500]. The original DR4 strain was of mixed background (129/SvJae, 129/OlaHsd, BALB/c, and C57BL/6). The cells can be used as a feeder layer to support the growth of embryonic stem (ES) cells and for the maintenance of ES cells in the undifferentiated state. 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
Propagation: MEF (DR4)細胞ATCC 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%
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37.0°C
Subculturing: Establishing cultures from frozen cells:
To insure the highest level of viability, pre-warm culture medium to 37.0°C before use.
Flasks do not need to be coated before plating MEFs.
Thaw the vial by gentle agitation in a 37.0°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.
Remove the vial from the water bath as soon as the contents are half way thawed (approximately 90 seconds), and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
Transfer the contents of the vial to a 15 ml centrifuge tube which contains 5.0 ml complete growth medium. Use an additional 1.0 ml of complete growth medium to rinse the vial and add this to the 15 ml tube. Adjust volume to 10.0 ml by adding 4.0 ml of complete growth medium.
Pellet the cells by centrifugation at 270 xg for 5 minutes. Remove and discard the supernatant.
Resuspend the cells in 10.0 ml fresh complete growth medium and transfer to a 75 sq. cm. flask.
Add 5.0 ml more fresh growth medium to flask.
Incubate 37.0°C in 5% CO2 in air atmosphere.
Subculturing Procedure:
Cells should be split when they reach confluency.
To insure the highest level of viability, warm culture medium, PBS and Trypsin-EDTA to 37.0°C before use.
Volumes used in this protocol are for 225 sq. cm. flasks; proportionally reduce or increase reagent volumes for culture flasks of other sizes.
Remove and discard culture medium.
Briefly rinse the cell layer with Ca++/Mg++ free phosphate-buffered saline (PBS) to remove all traces of serum, which contains trypsin inhibitor.
Add 5.0 ml of 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to flask and incubate for 1 minute. Gently tap the flask, and observe cells under an inverted microscope until cells detach (usually within 1 to 2 minutes).
Add 6.0 to 8.0 ml of complete growth medium and rinse surface of the flask to detach all cells. Gently pipette up and down to break up cell clumps.
Transfer cell suspension to a centrifuge tube and centrifuge at 270 xg for 5 minutes. MEF (DR4)細胞Remove and discard the supernatant.
Add 10 ml complete growth medium to cell pellet and resuspend the cells gently to create a single-cell suspension.
If necessary, add an appropriate volume of complete growth medium to cell suspension to seed cells at approximately 6 X10(3) cells/sq. cm.
Incubate cultures at 37.0°C in a 5% CO2 in air atmosphere.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:7 is recommended
Medium Renewal: Twice a week or when pH decreases
Preservation: Freeze medium: Complete growth medium supplemented with an additional 40% FBS and 10% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Related Products: Recommended medium (without the additional serum described under ATCC Medium): ATCC 30-2002
Recommended serum: ATCC 30-2020
0.25% (w/v) Trypsin - 0.53mM EDTA in Hank's BSS (w/o Ca++, Mg ++): ATCC 30-2101
Phosphate-buffered saline: ATCC SCRC-2201
Cell culture tested DMSO: ATCC 4-X
References: 89420: Tucker KL, et al. A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res. 25: 3745-3746, 1997. PubMed: 9278500
89421: Nagy A, et al. Manipulating The Mouse Embryo: A Laboratory Manual. Third Edition: Cold Spring Harbor Press; 2003.
