Chimeric Mouse Production by Microinjection

David A. Conner1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.7
DOI:  10.1002/0471142727.mb2307s53
Online Posting Date:  May, 2001
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Abstract

The culmination of the creation of a mutation in mouse embryonic stem (ES) cells is, commonly, the generation of a mouse line that can propagate the mutation. The ability to combine methods of homologous recombination in ES cells with blastocyst‐mediated transgenesis has resulted in an explosion of tailored mutant mouse strains. These animals provide research tools that are virtually impossible to create using other methodologies. This unit describes the methods necessary to generate chimeras, from injection of the ES cells into the blastocoel cavity of 3.5‐day‐old embryos through the implantation of the injected embryos into the foster mother. The resultant pups are true chimeras: their tissues are derived from both the host embryo and from the ES cells. If the ES cells are able to populate the germ line, the chimera can pass an altered gene to offspring, resulting in a new mouse strain in which all cells contain an altered gene.

     
 
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Table of Contents

  • Basic Protocol 1: Blastocyst Isolation
  • Basic Protocol 2: Blastocyst Injection
  • Basic Protocol 3: Uterine Transfers
  • Support Protocol 1: Preparation of Pseudopregnant Foster Mothers
  • Support Protocol 2: ES Cell Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Blastocyst Isolation

  Materials
  • C57BL/6 female mice (3 to 4 weeks old; e.g., Taconic)
  • 50 U/ml recipepregnant mare's serum (PMS; see recipe)
  • 50 U/ml recipehuman chorionic gonadotropin (HCG; see recipe)
  • C57BL/6 stud males (8 weeks to 6 months old; e.g., Taconic)
  • 95% ethanol
  • recipeInjection medium (see recipe)
  • Tuberculin syringe (1 ml, 26‐G, ⅜ in.)
  • Surgical equipment (thoroughly washed and sterilized by autoclaving or with ethanol; individual investigators may prefer slightly different instruments):
  •  The following are available from Biomedical Research Instruments (similar instruments are available from several suppliers):
  •  Scissors, large (4.5 in.)
  •  Scissors, small (3.5 in.)
  •  Scissors, iris (3 in.)
  •  Forceps, small (4 in.), curved
  •  Forceps, large (4 in.), curved
  •  Forceps, toothed (3.5 in.)
  • recipeBlunted 25‐G needle (see recipe) with 3, 5, or 10 ml syringe
  • Watch glass (Corning, 2.5 in., washed and sterilized with ethanol prior to use)
  • Dissecting microscope and fiber optic light source (stereo microscope, 0.8× to 4× zoom, with stand that allows illumination from above and below)
  • recipeEmbryo transfer pipet (see recipe)
  • Microdrops of injection medium under mineral oil (see recipe for recipemicrodrop cultures) in 35 × 10–mm petri dishes (Falcon)
  • Light white mineral oil (Sigma)

Basic Protocol 2: Blastocyst Injection

  Materials
  • recipeInjection medium (see recipe)
  • ES cell suspension (see protocol 5)
  • Blastocysts (see protocol 1)
  • Injection chamber (Fig. )
  • recipeInjection apparatus (see recipe)
  • Holding pipet (VacuTips, Eppendorf)
  • Injection pipet (Transfer Tips, Eppendorf)

Basic Protocol 3: Uterine Transfers

  Materials
  • Embryos (injected blastocysts; see protocol 2)
  • recipeInjection medium (see recipe)
  • 2.5‐day pseudopregnant female mice (see protocol 4)
  • recipe2.5% Avertin (see recipe)
  • 95% ethanol
  • Embryo transfer pipets (see recipe)
  • Modeling clay
  • Surgical equipment (thoroughly washed and sterilized by autoclaving or with ethanol; individual investigators may prefer slightly different instruments)
  •  The following are available from Biomedical Research Instruments (similar instruments are available from several suppliers):
  •  Scissors, large (4.5 in.)
  •  Scissors, small (3.5 in.)
  •  Scissors, iris (3 in.)
  •  Forceps, small (4 in.), curved
  •  Forceps, large (4 in.), curved
  •  Forceps, toothed (3.5 in.)
  •  Serrefine clamps
  • The following are available from Fisher (similar instruments are available from several suppliers)
  •  Wound clip applicator
  •  Wound clips (surgical staples)
  • Dissecting microscope and fiber optic light source (stereo microscope, 0.8× to 4× zoom, with stand that allows illumination from above and below)
  • 25‐G needle
  • Suture, 5‐0 Dexon II on T‐31 needle (e.g., Kendall Health Care)

Support Protocol 1: Preparation of Pseudopregnant Foster Mothers

  Materials
  • Vasectomized male mice (8 weeks to 16 months old; Taconic)
  • Female mice (8 weeks to 6 months old; Taconic)

Support Protocol 2: ES Cell Preparation

  Materials
  • recipeInjection medium (see recipe)
  • Hanks' balanced salt solution (HBSS; calcium‐ and magnesium‐free; unit 23.2)
  • Additional reagents and equipment for ES cell culture (unit 23.3)
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Figures

Videos

Literature Cited

   Bradley, A., Evans, M., Kaufman, M.H., and Robertson, E. 1984. Formation of germ‐line chimaeras from embryo‐derived teratocarcinoma cell lines. Nature 309:255‐256.
   Brinster, R.L. 1974. The effect of cells transferred into the mouse blastocyst on subsequent development. J. Exp. Med. 140:1049‐1056.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154‐156.
   Gardner, R.L. 1968. Mouse chimeras obtained by the injection of cells into the blastocyst. Nature 220:596‐597.
   Gossler, A., Doetschman, T., Korn, R., Serfling, E., and Kemler, R. 1986. Transgenesis by means of blastocyst‐derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA 83:9065‐9069.
   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, N.Y.
   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. USA 78:7634‐7636.
   Mince, B. and Illmensee, K. 1975. Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. Natl. Acad. Sci. U.S.A. 72:3585‐3599.
   Nagy, A. and Rossant, J. 1993. Production of completely ES cell‐derived fetuses. In Gene Targeting: A Practical Approach (A.L. Joyner, ed.). Oxford University Press, New York.
   Robertson, E., Bradley, A., Kuehn, M., and Evans, M. 1986. Germ‐line transmission of genes introduced into cultured pluripotential cells by a retroviral vector. Nature 323:445‐448.
   Rossant, J. and McBurney, M.W. 1983. Diploid teratocarcinoma cell lines differ in their ability to differentiate normally after blastocyst injection. Teratocarcinom Stem Cells, Cold Spring Harbor Conferences on Cell Proliferation 10:625‐633.
   Tarkowski, A.K. 1961. Mouse chimeras developed from fused eggs. Nature 190:857‐860.
Key References
   Hogan et al., 1994. See above.
  These two references, written by experts in the field, represent thorough compilations of transgenic methods.
   Papaioannou, V. and Johnson, R. 1993. Production of chimeras and genetically defined offspring from targeted ES cells. In Gene Targeting: A Practical Approach (A.L. Joyner, ed.) pp. 33‐61. IRL Press, Oxford.
Internet Resources
   http://www.biosupplynet.com
  Search this web site to obtain a current list of suppliers for materials and reagents used in the production of chimeras by blastocyst injection.
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