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Cell Biology
ATCC® Number: SCRC-1021™    Price: $649.00
Designations: EDJ#22
Depositors:  TJ Ley
Biosafety Level: 1
Shipped: frozen
Medium & Serum: See Propagation
Organism: Mus musculus (mouse)
Morphology: stem cell
Source: Organ:embryo
Cell Type: stem cell embryonic stem cell;
Strain:129 SvEv/tac
Tissue: Inner Cell Mass
Permits/Forms: 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.
Isolation: Isolation date: 2003
Cytogenetic Analysis: 40 XY, diploid
Age: embryo, blastocyst
Gender: male
Comments: This mouse ES cell line has been shown to be germline competent. 129 and, the more common, 129Sv are widely used mouse strains in gene targeting experiments. The embryonic stem cells derived from 129Sv are part of the genetic background of most mutants generated using homologous recombination. The 129/SvEv cell lines tested at early passages were found to contribute extensively to chimeras and produce germ-transmitting male chimeras. Furthermore, this cell line was able to maintain these characteristics after many passages in vitro. The 129SvEv ES cell line, EDJ 22, is derived from 129SvEV mice from Taconic. 129SvEv is a steel substrain with pigment white-bellied agouti. Taconic indirectly obtained 129/SvEvBrd and 129/SvEv-Gpilc, and crossed them to produce 129/SvETac. Inbreeding of 129/SvEvTac has been ongoing since 1992 and the substrain is now homozygous Gpilc. [57458] [16173166] [16173171]
Propagation: ATCC complete growth medium: ES-DMEM (ATCC SCRR-2010) supplemented with 2.0 mM L-Alanyl-L-Glutamine (ATCC 30-2115), 0.1 mM non-essential Amino Acids (ATCC 30-2116), 0.1 mM 2-mercaptoethanol (Invitrogen Life Technologies No. 21985), 1000 U/ml mouse leukemia inhibitory factor (LIF) (Chemicon No. ESG1107) and 15% fetal bovine serum (ATCC SCRR-30-2020).
Temperature: 37.0°C
Growth condition: feeder cells required.
Subculturing: Protocol: Establishing and maintaining your culture:To insure the highest level of viability, be sure to warm media to 37C before using it on the cells.
  1. Plate mitotically arrested MEFs (CF-1) (ATCCSCRC-1040)as a feeder layer at approximately 55,000 feeder cells/cm2 in complete medium for feeder cells. Refer to the product sheet for mitotically arrested MEF for detailed handling instructions. One hour before thawing the vial of ES cells, perform a 100% medium change using complete ES-DMEM (see below for recipe).
  2. Thaw the vial by gentle agitation in a 37C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 90 seconds).
  3. Remove the vial from the water bath before the contents are completely thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  4. Transfer the vial contents plus 5 ml of complete ES-DMEM to a 15 ml centrifuge tube. Use an additional 1 ml of media to rinse the vial and transfer the liquid to the 15 ml tube. Add 4 ml of complete ES-DMEM to bring the total volume to 10 ml.
  5. Spin the cells at 270 x g for 5 min. Aspirate the supernatant and resuspend the pellet in 2 ml of complete ES-DMEM.
  6. Add the 2 ml of cell suspension to the appropriate size flask containing feeder cells and fresh complete growth medium (see batch specific information). ES cells should be plated at a density of 30,000 to 50,000 cells/ cm2.
  7. Incubate the culture at 37C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1 to 2 days.

Subculturing Procedure: To insure the highest level of viability, be sure to warm media and Trypsin - EDTA to 37C before using it on the 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 every 1 to 2 days is recommended. Plating densities should range from 30,000 - 50,000 cells/ cm2. Note: If the colonies are close to or touching each other the culture is overgrown . Overgrowth will result in differentiation.
  1. Prepare enough flasks with MEFs as stated above in step #1.
  2. Aspirate the medium from the flask(s) with the ES cells.
  3. Wash with PBS (Ca+2/Mg+2-free, ATCCSCRR-2201).
  4. Add 3.0 ml of 0.25% (w/v) Trypsin - 0.53 mM EDTA solution (ATCC30-2101) to each T75 and place the flasks in the incubator. After one minute the ES colonies will dissociate and the cells will begin to detach from the flask.
  5. Dislodge the cells by gently tapping the side of the flask then wash the cells off with 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.
  6. Transfer the cell suspension to the appropriate size centrifuge tube.
  7. Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
  8. Resuspend in 30 to 50 ml of fresh culture medium, depending on the split ratio.
  9. Aspirate the medium from flasks containing feeders and replace it with cell suspension (15 ml/flask).
  10. Incubate the culture at 37C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1 to 2 days.

medium Renewal: Every day.
subcultivation Ratio: A subcultivation ratio of 1:4 to 1:7 is recommended.
Preservation: Storage temperature: liquid nitrogen vapor phase
Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO.
References: 57458: 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
16173130: Festing MF, et al. Revised nomenclature for strain 129 mice. Mamm. Genome. 10(8):836, 1999.[PubMed: 10430671]
16173166: Simpson EM, et al. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16(1):19-27, 1997. PubMed: 9140391
16173171: Threadgill DW, et al. Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain. Mamm. Genome. 8(6):390-393 1997. PubMed: 9166580
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