Embryonic Stem (ES) Cell Culture Basics

Jennifer V. Schmidt1

1 University of Illinois at Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 15.1
DOI:  10.1002/0471140856.tx1501s09
Online Posting Date:  November, 2001
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Abstract

This unit describes the procedures to grow and maintain embryonic stem (ES) cells in culture. Growth‐inactivated mouse embryo fibroblasts (MEF) are required as a feeder layer for the ES cells; their isolation and culture is described as are the additional protocols required for generating a mouse with a particular targeted gene electroporation, cloning and selection for homologous recombinants, transfection with Cre, and preparation of cells for injection.

     
 
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Table of Contents

  • Basic Protocol 1: ES Cell Culture
  • Support Protocol 1: Preparation of Primary Mouse Embryonic Fibroblast (pMEF) Cells
  • Support Protocol 2: Electroporation of ES Cells
  • Support Protocol 3: Transfection of CRE
  • Support Protocol 4: Preparation of Cells for Microinjection
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: ES Cell Culture

  Materials
  • 0.1% (w/v) gelatin
  • Mitomycin C–inactivated primary mouse embryonic fibroblast (pMEF) cells in a 1.5‐ml cryovial (see protocol 2)
  • pMEF medium, 37°C (see recipe)
  • ES cells in a 1.5‐ml cryovial (see protocol 3)
  • ES cell medium, 37°C (see recipe)
  • Trypsin/EDTA: 0.05% (w/v) trypsin/53 mM Na 4 EDTA (Invitrogen)
  • 10‐cm tissue culture dish or 6‐ or 96‐well tissue‐culture plate
  • 15‐ml culture tube
  • Sterile medium boats (96‐well plates)
  • Multichannel pipettor (96‐well plates)

Support Protocol 1: Preparation of Primary Mouse Embryonic Fibroblast (pMEF) Cells

  • Male transgenic mice homozygous for the neo gene (e.g., C57BL/6JTgN[pPGKneobpA]Ems, Jackson Laboratories)
  • Outbred nontransgenic female mice (preferably the same strain as the males)
  • PBS, fresh ( appendix 2A)
  • Freezing medium, 4°C (see recipe)
  • Mitomycin C medium (see recipe)
  • Dissecting equipment
  • Petri dish
  • Dissecting microscope
  • 6‐cm tissue culture dish
  • Razor or scalpel blades
  • 10‐ml serological pipets
  • 10‐ml syringe with 18‐G needle
  • 50‐ml centrifuge tube
  • 10‐cm tissue culture dish
  • 1.5‐ml cryovials
  • Additional reagents and equipment for isolating postimplantation embryos (unit 13.3)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 2: Electroporation of ES Cells

  • Targeting plasmid
  • H 2O, sterile
  • ES cell cultures (see protocol 1)
  • G418 medium or G418/ganciclovir medium (see reciperecipes)
  • Freezing medium (see recipe)
  • Electroporator (Bio‐Rad) and appropriate cuvettes with a 4‐mm gap
  • 15‐ml conical tube
  • 96‐well U‐ and flat‐bottom plates
  • Microscope with 4× objective
  • 200‐µl micropipettor and tips, sterile
  • Styrofoam box
  • Additional reagents and equipment for plasmid preparation by cesium chloride equilibrium centrifugation, enzyme digestion, agarose gel electrophoresis, phenol/chloroform/isoamyl extraction, and ethanol precipitation (see Table 97.80.4711A.3A.1 of appendix 3A), preparing 10‐cm feeder‐cell dishes (see protocol 2), and counting cells in a hemacytometer ( appendix 3B).

Support Protocol 3: Transfection of CRE

  • Circular Cre plasmid DNA
  • Targeted ES cells
  • H 2O, sterile
  • G418 medium (see recipe)
  • Electroporator and appropriate electroporation cuvettes with a 4‐mm gap
  • Microscope with 4× objective lens
  • 96‐well flat‐bottom plate
  • Additional reagents and equipment for plasmid preparation by cesium chloride equilibrium centrifugation (see Table 97.80.4711A.3A.1 of appendix 3A), counting cells in a hemocytometer ( appendix 3B), and Southern blotting (unit 3.6 and appendix 3A).

Support Protocol 4: Preparation of Cells for Microinjection

  Materials
  • ES cell cultures (see protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
   Bradley, A., Evans, M., Kaufman, M.H., and Robertson, E.J. 1984. Formation of germ‐line chimeras from embryo‐derived teratocarcinoma cell lines. Nature 309:255‐256.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154‐156.
   Hogan, B., Beddington, R., Constantini, F., and Lacy, E. 1994. Manipulating the Mouse Genome. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Robertson, E.J. 1997. Derivation and maintenance of embryonic stem cell cultures. In Methods in Molecular Biology, Vol. 75: Basic Cell Culture Protocols (J.W. Pollard and J.M. Walker, eds.) pp. 173‐184. Humana Press, Totowa, N.J.
   Simpson, E.M., Linder, C.C., Sargent, E.E., Davisson, M.T., Mobraaten, L.E., and Sharp, J.J. 1997. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nature Genet. 16:19‐27.
   Threadgill, D.W., Yee, D., Matin, A., Nadeau, J.H., and Magnuson, T. 1997. Genealogy of the 129 inbred strains: 129SvJ is a contaminated inbred strain. Mammal. Genome 8:390‐393.
Key References
   Hogan et al., 1994. See above.
   The above reference is an excellent manual that describes both theory and practice of gene targeting in embryonic stem cells.
   Kuhn, R. and Schwenk, F. 1997. Advances in gene targeting methods. Curr. Opin. Immunol. 9:183‐188.
   Another well‐written review of gene targeting methods.
   Muller, U. 1999. Ten years of gene targeting: Targeted mouse mutants, from vector design to phenotype analysis. Mech. Dev. 82:3‐21.
   An extremely comprehensive and up‐to‐date review covering all aspects of gene targeting.
   Robertson, 1997. See above.
   The above reference is an excellent manual that describes both the theory and practice of gene targeting in embryonic stem cells.
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