Aggregation Chimeras (ES Cell–Embryo)

Corrinne G. Lobe1, Caiying Guo1

1 Sunnybrook and Women's College Health, Sciences Centre and the University of Toronto, Toronto, Canada
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 15.3
DOI:  10.1002/0471140856.tx1503s10
Online Posting Date:  February, 2002
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Abstract

One approach for generating transgenic mice from ES cell lines is to aggregate ES cells with morula‐stage embryos to generate chimeric mice. The chimeras are then bred to generate transgenic offspring. This method offers a simpler and less expensive alternative to the method of ES cell injection into blastocyst embryos.

     
 
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Table of Contents

  • Basic Protocol 1: Preparing ES Cell–Embryo Aggregation Chimeras
  • Support Protocol 1: Mouse Husbandary
  • Support Protocol 2: Making Aggregation Plates
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparing ES Cell–Embryo Aggregation Chimeras

  Materials
  • M2 medium (see recipe)
  • KSOM medium (see recipe)
  • 2.5‐dpc superovulated donor mice (e.g., CD‐1; see protocol 2)
  • Acid Tyrode's solution (Sigma)
  • ES cells (unit 15.1), trypsinized
  • Light mineral oil, embryo tested
  • 2.5‐ or 3.5‐dpc pseudopregnant recipient mice (see protocol 2)
  • 1.25% (w/v) avertin (see recipe)
  • 70% (v/v) ethanol in a squeeze bottle
  • 1‐ and 5‐ml syringes with 26‐G needles
  • 100 × 15–mm petri dish, sterile
  • Surgical instruments: 2 straight or curved forceps with serrated tips, small straight or curved scissors, no. 5 forceps (Dumont), forceps with 1 × 2 teeth (i.e., “rat's tooth”), sharp forceps, serrefine clamps (e.g., Fine Scientific Tools), and suture clips and applier (Fine Scientific Tools)
  • 1‐ml syringe with flushing needle (see recipe)
  • Dissecting microscope
  • Finger pipet (see recipe)
  • Prepared aggregation plate with depressions (see protocol 3)
  • Embryo‐transfer pipet (see recipe) and appropriate mouth piece
  • 28‐G needle
  • Lamp
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic techniques should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Mouse Husbandary

  • 35 × 10–mm tissue culture dishes, sterile
  • Aggregation (i.e., darning) needle (BLS)
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Figures

Videos

Literature Cited

Literature Cited
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   Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. 1985. The in vitro development of blastocyst‐derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morph. 87:27‐45.
   Doetschman, T., Maeda, N., and Smithies, O. 1988. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 85:8583‐8587.
   Friedrich, G. and Soriano, P. 1991. Promoter traps in embryonic stem cells: A genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5:1513‐1523.
   Lobe, C.G., Koop, K.E., Kreppner, W., Lomeli, H., Gertsenstein, M., and Nagy, A. 1999. Z/AP, a double reporter for cre‐mediated recombination. Dev. Biol. 208:281‐292.
   Nagy, A., Rossant, J., Nagy, R., Abramow‐Newerly, W., and Roder, J. 1993. Derivation of completely cell culture‐derived mice from early‐passage embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 90:8424‐8428.
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   Skarnes, W.C., Auerbach, B.A., and Joyner, A.L. 1992. A gene trap approach in mouse embryonic stem cells: The lacZ reporter is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice. Genes Dev. 6:903‐918.
   Stewart, C.L. 1993. Production of chimeras between embryonic stem cells and embryos. Methods Enzymol. 225:823‐855.
   Thomas, K. and Capecchi, M. 1987. Site‐directed mutagenesis by gene targeting in mouse embryo‐derived stem cells. Cell 51:503‐512.
   Wood, S.A., Allen, N.D., Rossant, J., Auerbach, A., and Nagy, A. 1993. Non‐injection methods for the production of embryonic stem cell‐embryo chimeras. Nature 365:87‐89.
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