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RW.4
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TJ Ley |
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Biosafety Level: |
1
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frozen
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Medium & Serum:
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See Propagation
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adherent |
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Mus musculus (mouse)
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spherical colony
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Organ: embryo, blastocyst
Tissue: inner cell mass
Cell Type: embryonic stem cell |
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In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.
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Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact the ATCCOffice of Licensing and Business Development at licensing@ATCC.org or 703 365 2773.
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Gene knock-out - RW.4 cells have been used to create a number of different gene knock-out mice. [90419] [16173329]
Gene knock-in - RW.4 ES cells were used to create a knock-in mouse subline for use as a model of acute promyelocytic leukemia (APL). [16173330]
Differentiation - RW.4 ES cells can be induced by staurosporine (STS) to differentiate into neurons and astrocytes from embryoid bodies. [16173319] [16173331]
ESC Proliferation - RW.4 cells have been used to study the effects of statins, cholesterol-lowering drugs, on ESC self-renewal, proliferation and differentiation. |
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embryo, blastocyst |
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male |
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ATCC complete growth medium: The base medium for this cell line is ES-DMEM (ATCC Cat. No.SCRR-2010). To make the complete growth medium, add the following components to the base medium: - 15% fetal bovine serum (ATCC Cat. No.SCRR-30-2020)
- 2.0 mM L-Alanyl-L-Glutamine (ATCC Cat. No. 30-2115)
- 0.1 mM MEM non-essential amino acids (ATCC Cat. No. 30-2116)
- 0.02% bovine serum albumin
- 0.1 mM 2-mercaptoethanol (InvitrogenCat. No.21985)
- 1000 U/ml mouse leukemia inhibitory factor (LIF) (Chemicon Cat. No. SG1107)
This medium is formulated for use with a 5% CO2 in air atmosphere. Standard DMEM formulations contain 3.7 g/L sodium bicarbonate. A 10% CO2 in air atmosphere is then recommended.
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37.0°C |
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ATCCrecommends culturing RW.4 on mouse embryonic fibroblasts (MEFs) that have been mitotically arrested by either irradiation or treatment with mitomycin-C. RW.4 cells have been cultured on irradiated or mitomycin-C treated MEF (CF-1) (ATCCSCRC-1040.1 or SCRC-1040.2a).Feeder cells should be initiated 24-48 hours prior to inoculating with embryonic stem (ES) cells layer at approximately 5.5 X10(4) feeder cells/cm2.
Protocol: Volumes used in this protocol are for 75 sq cm flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Subculture the cells every 1 to 2 days. If the colonies are close to or touching each other the culture is overgrown. Overgrowth will result in differentiation. Make sure that sufficient number of flasks pre-plated with MEF feeder layers are prepared in advance to support frequent passage of the ES cells.Pre-warm media and Trypsin/EDTA to 37ºC before adding to cells.- Remove and discard culture medium.
- Briefly rinse the cell layer with Ca++/Mg++ free Phosphate Buffered Saline (ATCCSCRR-2201 to remove all traces of serum which contains trypsin inhibitor.
- Add 2.0 to 3.0 ml of 0.25% (w/v) Trypsin-0.53 mM EDTA (ATCC® 30-2101) solution to flask and observe cells under an inverted microscope . After about one minute the ES colonies will dissociate and all cells will detach.
- Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting several times with a 10 ml pipette in order to dissociate the cells into a single-cell suspension.
- Transfer cell suspension to a centrifuge tube and spin at approximately 125 X g for 5 to 10 minutes.
- Discard supernatantand resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to culture vessels with feeder layer. An inoculum of 3 X 10(4) to 5 X 10(4) viable cells/sq. cm is recommended.
- Incubate cultures at 37C.
Interval: every 1 to 2 days
Subcultivation Ratio: 1:4 to 1:6
Medium Renewal: Every day |
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Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO.
Storage temperature: liquid nitrogen vapor phase |
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recommended serum:ATCC 30-2020
MEM Non-Essential Amino Acid Solution, 100x:ATCC 30-2116
Recommended medium (without the additional supplements or serum described under ATCC Medium):ATCC SCRR-2010
L-Alanyl-L-Glutamine Solution, 200 mM:ATCC 30-2115 |
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90419: Hug BA, et al. Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3. Mol. Cell. Biol. 16: 2906-2912, 1996. PubMed: 8649401
90420: Matise MP, et al. Production of targeted Embryonic Stem Cell Clones. In AL Joyner (Ed.), Gene Targeting: A Practical Approach. Oxford University Press, Oxford, p. 101-132, 1999.
16173319: Fabricius D, Bonde S, Zavazava N. Induction of stable mixed chimerism by embryonic stem cells requires functional Fas/FasL engagement. Transplantation 79(9): 1040-1044, 2005. PubMed: 15880040
16173329: Conte D, et al. Inhibitor of apoptosis protein cIAP2 is essential for lipopolysaccharide-induced macrophage survival. Mol. Cell Biol. 26(2): 699-708, 2006. PubMed: 16382159
16173330: Westervelt P, et al. High-penetrance mouse model of acute promyelocytic leukemia with very low levels of PML-RARalpha expression. Blood 102(5): 1857-1865, 2003. PubMed: 12750176
16173331: Schumacher A, et al. Staurosporine is a potent activator of neuronal, glial, and "CNS stem cell-like" neurosphere differentiation in murine embryonic stem cells. Mol. Cell Neurosci. 23: 669-680, 2003. PubMed: 12932446
16173332: Lee MH, et al. Simvastatin suppresses self-renewal of mouse embryonic stem cells by inhibiting RhoA geranylgeranylation. Stem Cells 25: 1654-1663, 2007. PubMed: 17464088 |
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