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EDJ#22; Mouse ES Item ID: ATCSCRC1021

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Organism Mus musculus, mouse
Tissue inner cell mass
Cell Type embryonic stem cell
Product Format frozen
Morphology stem cell
Biosafety Level 1 [Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office (2007). The entire text is available online at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.]

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Age embryo
Gender Male
Strain 129S5/SvEvTac
Storage Conditions liquid nitrogen vapor phase
Karyotype 40 XY, diploid
Derivation 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.ref
Clinical Data 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. RefThreadgill 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  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. RefAuerbach 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
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

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

Complete growth medium supplemented with an additional 10% FBS and 10% DMSO. Make immediately prior to use. Keep at 4°C.
Culture Conditions Temperature: 37°C Growth condition: feeder cells required.
Name of Depositor TJ Ley
Year of Origin 2003
References

Festing MF, et al. Revised nomenclature for strain 129 mice. Mamm. Genome. 10(8):836, 1999.[PubMed: 10430671]

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

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

Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988

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