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.

This mouse ES cell line has been shown to be germline competent. 129Sv/J is a parental substrain of 129 strains and has a white-bellied, light chinchilla or albino coat color with pink eyed appearance. This strain is homozygous for Cdh23^ahl, the age-related hearing loss 1 mutation, resulting in progressive hearing loss with onset prior to 3 months of age (http://jaxmice.jax.org/strain/000691.html).

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.

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

Gossler A, et al. Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA 83: 9065-9069, 1986. PubMed: 3024164

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

Stevens LC. A new inbred subline of mice (129-terSv) with a high incidence of spontaneous congenital testicular teratomas. J. Natl. Cancer. Inst. 50(1): 235-242, 1973. PubMed: 4692863

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

Jackson Laboratory

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

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.