7AC5/EYFP (ATCC® SCRC-1033)

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

Permits and Restrictions

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Organism Mus musculus, mouse
Tissue inner cell mass
Cell Type embryonic stem cell
Product Format frozen
Culture Properties adherent
Biosafety Level 1
Age embryo
Gender male
Strain 129X1 x 129S1
Applications
SCRC-1033 has been shown to be germline competent, stains positive for pluripotency markers and alkaline phosphatase activity, and survives puromycin selection at concentrations of up to 0.004 mg/mL.
Storage Conditions liquid nitrogen vapor phase
Clinical Data
male
Comments
7AC5/EYFP is a yellow fluorescent variant of R1 ES cells (ATCC SCRC-1011). The fluorescent variant was generated by the random integration of EYFP into R1 ES cells using co-electroporation with a circular selectable marker containing vector pPGK Puro. The vector is driven by a CMV immediate early enhancer coupled to the chicken beta-actin promoter and first intron. SCRC-1033 has been shown to be germline competent, stains positive for pluripotency markers and alkaline phosphatase activity, and survives puromycin selection at concentrations of up to 0.004 mg/mL.
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 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
Growth Conditions: SCRC-1033 should be grown on Mitomycin C treated or irradiated mouse embryonic fibroblasts in the presence of complete growth medium in order to maintain them in an undifferentiated state.
Name of Depositor A Nagy
References

Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999

Hadjantonakis AK, et al. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76: 79-90, 1998. PubMed: 9867352

Hadjantonakis AK, Nagy A. FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis 27: 95-98, 2000. PubMed: 10951501

Hadjantonakis AK, Nagy A. The color of mice: in the light of GFP-variant reporters. Histochem. Cell Biol. 115: 49-58, 2001. PubMed: 11219608

Hadjantonakis AK, et al. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol. 2: 11, 2002. PubMed: 12079497

Basic Documentation
Restrictions

This product's use is governed by the Limited Use License. For information on purchasing a license to use this product for research (for-profit entities) or commercial purposes (any entity) other than those permitted in the Limited Use License, contact the Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California 92008. Phone (760) 603-7200 or outlicensing@lifetech.com
In addition, distribution of this cell line is further governed by Mt. Sinai Hospital MTA, Apx. A. For instructions on how to proceed, please contact ATCC's Office of Corporate Development at licensing@atcc.org.

References

Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999

Hadjantonakis AK, et al. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76: 79-90, 1998. PubMed: 9867352

Hadjantonakis AK, Nagy A. FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis 27: 95-98, 2000. PubMed: 10951501

Hadjantonakis AK, Nagy A. The color of mice: in the light of GFP-variant reporters. Histochem. Cell Biol. 115: 49-58, 2001. PubMed: 11219608

Hadjantonakis AK, et al. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol. 2: 11, 2002. PubMed: 12079497