Mouse Embryonic Stem (ES) Cell Culture

David A. Conner1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.3
DOI:  10.1002/0471142727.mb2303s51
Online Posting Date:  May, 2001
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Abstract

Mouse embryonic stem (ES) cells are used to generate mouse mutants by gene targeting and blastocyst‐mediated transgenesis. ES cells must be cultured under conditions that prevent differentiation to maintain their ability to transmit altered alleles by contributing to the germ line. This unit describes one of the most common methods for culturing ES cells using mouse embryo fibroblast (MEF) feeder layers and recombinant leukemia inhibitory factor (LIF) to prevent differentiation.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • recipe0.1% (w/v) gelatin solution (see recipe)
  • Mitotically inactive mouse embryo fibroblasts (MEFs; unit 23.2)
  • recipeES cell medium (see recipe)
  • Embryonic stem (ES) cells (http://www.biosupplynet.com)
  • Ca2+‐ and Mg2+‐free HBSS (unit 23.2)
  • recipeDPBS/EDTA solution (see recipe)
  • recipeTrypsin/EDTA solution (see recipe)
  • Freezing medium (unit 23.2)
  • Cryovials
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3F)
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Figures

Videos

Literature Cited

   Coté, R. 2000. Assessing and controlling microbial contamination in cell cultures. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott‐Schwartz, and K.M. Yamada, eds.) pp. 1.5.1‐1.5.18. John Wiley & Sons, New York.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154‐156.
   Martin, G.R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. U.S.A. 78:7634‐7636.
   Smith, A.G. and Hooper, M.L. 1987. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev. Biol. 121:1‐9.
   Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.G., Moreau, J., Stahl, M., and Rogers, D. 1988. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688‐690.
   Wurst, W. and Joyner, A.L. 1993. Production of targeted embryonic stem cell clones. In Gene Targeting: A Practical Approach (A.L. Joyner, ed.) pp. 33‐61. IRL Press, Oxford.
Key References
   Hogan, B., Beddington, R., Constantini, F., and Lacy, E. 1994. Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  These three references, written by experts in the field, represent the best compilations of ES cell culture methods to date.
   Robertson, E.J. 1987. Embryo‐derived stem cell lines. In Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (E.J. Robertson, ed.) pp. 71‐112. IRL Press, Oxford.
   Wurst and Joyner, 1993. See above.
Internet Resources
   www.biosupplynet.com
  Search this Web site for “embryonic stem cell reagents” to obtain a current list of suppliers that provide medium and ES cells.
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