RW.4 (ATCC® SCRC-1018)

Organism: Mus musculus, mouse  /  Cell Type: embryonic stem cell  /  Tissue: inner cell mass  / 

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Organism Mus musculus, mouse
Tissue inner cell mass
Cell Type embryonic stem cell
Product Format frozen
Morphology spherical colony
Culture Properties adherent
Biosafety Level 1
Age embryo
Gender male
Strain 129X1/SvJ
Applications
Gene knock-out - RW.4 cells have been used to create a number of different gene knock-out mice.
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).
Differentiation - RW.4 ES cells can be induced by staurosporine (STS) to differentiate into neurons and astrocytes from embryoid bodies.
ESC Proliferation - RW.4 cells have been used to study the effects of statins, cholesterol-lowering drugs, on ESC self-renewal, proliferation and differentiation.
Storage Conditions liquid nitrogen vapor phase
Clinical Data
male
Comments
The cells stain positive for pluripotency markers and alkaline phosphatase activity and RW.4 has been shown to be germline competent.
Complete Growth Medium Grow ES cells in Mouse ES Cell Basal Medium (ATCC SCRR-2011) that has been supplemented with the following components:
1. 0.1 mM 2-mercaptoethanol (Life Technologies Cat. No. 21985-023)
2. 1,000 U/mL mouse leukemia inhibitory factor (LIF) (Millipore Cat. No. ESG1107)
3. 10% to 15% ES-Cell Qualified FBS (ATCC® SCRR-30-2020) or an ES cell qualified serum replacement
Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C.
Complete Growth Medium
Grow ES cells in Mouse ES Cell Basal Medium (ATCC SCRR-2011) that has been supplemented with the following components:
0.1 mM 2-mercaptoethanol (Invitrogen Cat. No. 21985)
1,000 U/ml mouse leukemia inhibitory factor (LIF) (EMD Millipore Cat. No. ESG1107)

15% FBS, ES Cell Qualified (ATCC SCRR-30-2020)
Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C. This medium is formulated for use with a 5% CO2 in air atmosphere.
Subculturing Subculturing Procedure

Note: To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.

Feeder Cell Preparation for Subcultures

  1. Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.
  2. One hour before subculturing the ES cells, perform a 100% medium change for the MEFs using complete growth medium for ES cells.

Dissociation and Transfer of ES Cells

  1. Aspirate the medium from the flask(s) containing ES cells.
  2. Wash with PBS Ca+2/Mg+2-free (ATCC® SCRR-2201).
  3. Add 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC® 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.
  4. Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.
  5. Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
  6. Resuspend in enough complete growth medium for ES cells to reseed new vessels at the desired split ratio (i.e. a split ratio of 1:4 to 1:7 is recommended). Perform a cell count to determine the total number of cells. ES cells should be plated at a density of 30,000 – 50,000 cells/ cm2.
  7. Add separate aliquots of the cell suspension to the appropriate size flask containing feeder cells and add an appropriate volume of fresh complete growth medium for ES cells to each vessel.
  8. Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1-2 days.
Cryopreservation
Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO.
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Name of Depositor TJ Ley
References

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

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.

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

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

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

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

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

Basic Documentation
Restrictions

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

References

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

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.

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

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

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

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

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