RW.4細胞
器官來源： 胚胎
細胞類型： 其他細胞類型
年限： embryo, blastocyst
是否是腫瘤細胞： 0
物種來源： 小鼠
ATCC Number： SCRC-1018?
生長狀態： 貼壁生長
運輸方式： 凍存運輸
數量： 大量
細胞形態： 球形
組織來源： inner cell mass
Designations: RW.4
Depositors: TJ Ley
RW.4細胞Biosafety Level: 1
Shipped: frozen
Medium & Serum: See Propagation
Growth Properties: adherent
Organism: Mus musculus
Morphology: spherical colony
Source: Organ: embryo, blastocyst
Tissue: inner cell mass
Cell Type: embryonic stem cell
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Applications: 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]
RW.4細胞ESC Proliferation - RW.4 cells have been used to study the effects of statins, cholesterol-lowering drugs, on ESC self-renewal, proliferation and differentiation.
Age: embryo, blastocyst
Gender: male
Comments: The cells stain positive for pluripotency markers and alkaline phosphatase activity and RW.4 has been shown to be germline competent.
Propagation: ATCC complete growth medium: Mouse ES Cell Basal Medium
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37.0°C
Subculturing: ATCC recommends 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) (ATCC SCRC-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. RW.4細胞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 37°C before adding to cells.
Remove and discard culture medium.
Briefly rinse the cell layer with Ca++/Mg++ free Phosphate Buffered Saline (ATCC SCRR-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 104 to 5 X 104 viable cells/cm2 is recommended.
Incubate cultures at 37°C.
Interval: every 1 to 2 days
Subcultivation Ratio: 1:4 to 1:6
Medium Renewal: Every day
Preservation: Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO.
Storage temperature: liquid nitrogen vapor phase
Related Products: 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-2011
L-Alanyl-L-Glutamine Solution, 200 mM:ATCC 30-2115
References: 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. RW.4細胞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
