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The cells spontaneously differentiate into embryonic structures in the absence of a feeder layer or conditioned medium. They can be injected back into blastocysts and contribute to the germline.

Undifferentiated cells can be genetically modified by gene targeting techniques.

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note: The cells can be maintained in the undifferentiated state by frequent subculture on feeder layers of mitomycin C (MITC) treated (0.01 mg/mL for 90 minutes) primary mouse embryonic fibroblasts or STO cells (see ATCC CRL-1503, STO or ATCC 56-X.2, MITC-STO cells).

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add fresh medium, aspirate and dispense onto fresh feeder layer cultures.
6. Incubate cultures at 37°C.

Doetschman TC, et al. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87: 27-45, 1985. PubMed: 3897439

Williams RL, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336: 684-687, 1988. PubMed: 3143916

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

Doetschman T, et al. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc. Natl. Acad. Sci. USA 85: 8583-8587, 1988. PubMed: 3186749

Chambon P, et al. Genetically engineered mice containing alterations in the genes encoding retinoic acid receptor proteins. US Patent 6,486,381 dated Nov 26 2002