C57BL/6 (ATCC® SCRC-1002™)

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

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These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Organism Mus musculus, mouse
Tissue embryo; inner cell mass
Cell Type embryonic stem cell
Product Format frozen
Morphology spherical colony
Culture Properties Adherent
Biosafety Level 1
Age embryo
Strain C57BL/6
Storage Conditions Liquid nitrogen vapor phase
Derivation
The clonal embryonic stem cell line #693 ES C57BL/6 was derived from a strain C57BL/6J (B6) mouse blastocyst [PubMed: 11730008].
Comments
Coat-color chimera production was high using c2J blastocysts while FVB blastocysts produced a low number of chimeras [PubMed: 11730008].
The ES cells were shown to populate the germ line of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyrc-2J (c2J).
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.
Subculturing 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
Liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Name of Depositor JC Conover, B Knowles
References

Brook FA, et al. The derivation of highly germline-competent embryonic stem cells containing NOD-derived genome. Diabetes. 52:205-208, 2003. PubMed: 12502514

Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA. 94: 5709-5712, 1997. PubMed: 9159137

Schuster-Gossler K, et al. Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines. BioTechniques 31: 1022-1024, 1026, 2001. PubMed: 11730008

Auerbach W, et al. Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines. BioTechniques 29: 1024-1028, 1030, 1032, 2000. PubMed: 11084865

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.

Lemckert FA, et al. Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro. Nucleic Acids Res. 25: 917-918, 1997. PubMed: 9016649

Ledermann B, Burki K. Establishment of a germ-line competent C57BL/6 embryonic stem cell line. Exp. Cell Res. 197: 254-258, 1991. PubMed: 1959560

Permits Notice: Necessary Permits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Product Sheet
Product Sheet
Certificate of Analysis
Certificate of Analysis
MSDS
MSDS
Restrictions Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact ATCC's Office of Licensing and Business Development at licensing@atcc.org or 703 365 2773.
References

Brook FA, et al. The derivation of highly germline-competent embryonic stem cells containing NOD-derived genome. Diabetes. 52:205-208, 2003. PubMed: 12502514

Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA. 94: 5709-5712, 1997. PubMed: 9159137

Schuster-Gossler K, et al. Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines. BioTechniques 31: 1022-1024, 1026, 2001. PubMed: 11730008

Auerbach W, et al. Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines. BioTechniques 29: 1024-1028, 1030, 1032, 2000. PubMed: 11084865

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.

Lemckert FA, et al. Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro. Nucleic Acids Res. 25: 917-918, 1997. PubMed: 9016649

Ledermann B, Burki K. Establishment of a germ-line competent C57BL/6 embryonic stem cell line. Exp. Cell Res. 197: 254-258, 1991. PubMed: 1959560