| 產(chǎn)品名稱 |
MEF (DR4) |
| 商品貨號 |
B207260 |
| Organism |
Mus musculus, mouse |
| Tissue |
Embryo |
| Cell Type |
Fibroblast |
| Product Format |
frozen |
| Morphology |
fibroblast |
| Culture Properties |
Adherent |
| Biosafety Level |
1
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. |
| 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.
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| 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).
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| 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.
-
Remove and discard culture medium.
- 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.
- 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.
- 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.
- Transfer all cell suspension into a centrifuge tube and centrifuge at 270 xg for 5 minutes.
- Remove and discard the supernatant.
- Add complete growth medium to the cell pellet and with 10 mL pipette gently resuspend the cells gently to create a single-cell suspension.
- Adjust volume as needed to seed vessels at approximately 6 X103 cells/cm2.
- 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.
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