Transgenic Mouse Production By Zygote Injection

David A. Conner1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.9
DOI:  10.1002/0471142727.mb2309s68
Online Posting Date:  November, 2004
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Abstract

This unit describes methods for the production of transgenic mice by injection of DNA into zygotes, including fertilized-egg isolation, zygote injection, and oviduct reimplantation. Methods for the preparation of plasmid and BAC DNA suitable for microinjection are also presented.

Keywords: transgenic; transgene; zygote; pronuclear injection

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Isolation of Fertilized Eggs
  • Basic Protocol 2: Injection of Zygotes
  • Basic Protocol 3: Transfer of Oviducts to Foster Mothers
  • Support Protocol 1: Preparation of Plasmid-Based Transgene DNA
  • Support Protocol 2: Preparation of BAC-Based Transgene DNA
  • Support Protocol 3: Preparation of Equipment for Surgery, Embryo Transfer, and Egg Injection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Fertilized Eggs

 Materials
  • 50 U/ml PMS (see recipe)
  • 3- to 4-week-old FVB female mice (Taconic or The Jackson Laboratory)
  • 50 U/ml HCG
  • 8-week- to 1-year-old fertile stud male mice, individually caged
  • 95% ethanol
  • M2 medium (Sigma or Specialty Media) containing 100 U/ml penicillin and 100 µg/ml streptomycin (add antibiotics just before use)
  • 1 mg/ml hyaluronidase (Sigma H-3884) in M2 medium
  • M16 microdrop cultures (see recipe)
  • 1-ml, -in. tuberculin syringes with 26-G needles
  • Surgical equipment (see Support Protocol 3):
    • Scissors
    • Fine forceps
    • Iris scissors
  • 35 × 10–mm and 60 × 10–mm petri dishes
  • Dissecting microscope: stereomicroscope with fiber-optic light source, 0.8× to 4× zoom, and a stand that allows illumination from above and below
  • Embryo transfer pipet (see Support Protocol 3)
  • 37°C, 5% CO2 incubator
  • Additional reagents and equipment for preparing foster mothers (UNIT 23.7)

Basic Protocol 2: Injection of Zygotes

 Materials
  • M2 medium (Sigma or Specialty Media) containing 100 U/ml penicillin and 100 µg/ml streptomycin (add antibiotics just before use)
  • Embryo-tested mineral oil (Sigma)
  • Zygotes (see Basic Protocol 1)
  • M16 medium (Sigma or Specialty Media) containing 100 U/ml penicillin and 100 µg/ml streptomycin
  • Culture slide with a single ~18-mm-diameter 0.8-mm deep depression (i.e., depression slide, VWR)
  • Injection apparatus with holding pipet and injection pipet loaded with DNA solution (see Support Protocol 3)
  • Embryo transfer pipet (see Support Protocol 3)
  • 37°C, 5% CO2 incubator
  • Transgene in injection buffer (see Support Protocols 1 or 2)

Basic Protocol 3: Transfer of Oviducts to Foster Mothers

 Materials
  • M2 medium (Sigma or Specialty Media) containing 100 U/ml penicillin and 100 µg/ml streptomycin
  • Injected zygotes (see Basic Protocol 2)
  • Modeling clay (VWR, WL6852)
  • 0.5-day-p.c. Pseudopregnant females
  • 2.5% avertin (see recipe)
  • 70% ethanol
  • Analgesics per institutional requirements
  • Embryo transfer pipets (Support Protocol 3)
  • 1-ml, -in. tuberculin syringes with 26-G needles
  • Surgical equipment and supplies (Support Protocol 3):
    • Electric clippers
    • Scissors
    • Toothed and blunt forceps
    • Iris scissors
    • Serrefine clamp
    • Sutures
    • Surgical staples
    • Dissecting microscope with overhead illumination
  • 30-G needle (VWR)
  • Heating element (see Support Protocol 3)
  • Fresh mouse cages

NOTE: Follow institutional animal care guidelines for survival surgery.

NOTE: Trying both oviduct puncture and infundibular transfer is recommended. Use whichever is least difficult.

Support Protocol 1: Preparation of Plasmid-Based Transgene DNA

 Additional Materials
  • Plasmid DNA containing transgene construct (UNIT 1.7)
  • Low-melting-point agarose (e.g., Roche)
  • TAE or TBE (APPENDIX 2A)
  • Elutip low-salt solution (see recipe), 42°C
  • Elutip-d column (Schleicher & Schuell)
  • Elutip high-salt solution (see recipe)
  • 70% ethanol
  • Plasmid injection buffer (see recipe)
  • 42° and 65°C water baths
  • 5- and 10-ml syringes
  • Additional reagents and equipment for restriction enzyme analysis (UNIT 3.1), agarose gel electrophoresis (UNITS 2.5A & 2.6), and ethanol precipitation (UNIT 2.1A)

Support Protocol 2: Preparation of BAC-Based Transgene DNA

 Materials
  • BAC-transfected bacteria (e.g., UNITS 1.8 & 5.9)
  • LB medium (UNIT 1.1)
  • Nucleobond Plasmid EF Maxi Kit (BD Biosciences):
    • S1-EF buffer
    • RNase A
    • S3-EF buffer
    • N3-EF buffer
    • N5-EF buffer
    • Nucleobond AX 500 column
    • Filter
  • 2-propanol
  • 70% ethanol
  • BAC injection buffer (see recipe)
  • Additional reagents and solutions for agarose gel electrophoresis (UNIT 2.5A)

NOTE: Mega or giga kits can be used to process a larger volume of overnight culture. Remember to adjust the buffer volumes accordingly if larger cultures are used.
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Figures

  •  FigureFigure 23.9.1 Uterine horns, oviduct, and ovaries during various stages of fertilized egg isolation. (A) Photograph of the uterine horns with oviduct, ovary, and fat pad still attached. Excision as a unit may be easier for the beginner than removal of the oviducts and ovaries alone. Note that the oviduct and ovary on the left side has been removed. (B) Diagram of similar structure seen in (A), with suggested cut sites to separate the oviducts and ovaries from the uterus. Grasp the ovarian fat pad with forceps to move the oviduct and ovary.
    View Image
  •  FigureFigure 23.9.2 Oviduct and ovary after removal of the uterine horn. Note the location of the bursa, a thin transparent membrane that covers the gap between the oviduct and ovary. (A) The line indicates the cut site to separate the oviduct from the ovaries. (B) Diagram of the image seen in (A). Note the swollen region of the oviduct, the ampulla, near the ovarian end of the oviduct, which is often hidden under the folds of the oviducts. The dotted line indicates where to cut to separate the oviducts from the ovary.
    View Image
  •  FigureFigure 23.9.3 Diagram of the isolated oviducts. Tear the swollen ampulla of the oviduct with fine forceps as shown to release the cumulus mass containing fertilized eggs and follicle cells.
    View Image
  •  FigureFigure 23.9.4 Zygotes in the cumulus mass and after final isolation. (A) Photograph of the cumulus mass immediately after release from the swollen ampulla. Multiple fertilized eggs are visible in a mass of cumulus cells. (B) Photograph of zygotes after hyaluronidase treatment and subsequent transfer to a new microdrop. This is a mixture of fertilized and unfertilized eggs. Note the eggs with two pronuclei indicating fertilization. These are the eggs that are candidates for injection. The polar body is also visible in most eggs.
    View Image
  •  FigureFigure 23.9.5 Injection chamber and pipet alignment. (A) Diagram illustrating top and side views of an injection chamber using a depression slide. (B) The upper diagram illustrates one method for bringing a pipet tip into alignment. Under low magnification, use the course controls to place the pipet in the 6 o'clock position with the top just at the surface of the microdrop. While looking through the microscope, focused on the zygotes in the bottom of the depression, move the pipet tip toward the 12 o'clock position. (The pipet will appear as an out-of-focus blur as it crosses the field of view.) Stop when the tip is centered in the field. Always know where the bottom of the chamber and the pipet tip are when moving the tip up and down, to avoid breakage. The middle diagram illustrates the correct orientation of both pipets for injection viewed from the side. The angle of the tips should be as oblique as possible while allowing the tip to reach the bottom of the chamber without the barrel of the pipet touching the rim of the chamber. The bottom diagram illustrates the correct orientation of both pipets for injection viewed from the top. Note that the pipet shafts are both oriented along the same straight line.
    View Image
  •  FigureFigure 23.9.6 Alignment and injection. (A) Photograph of zygote attached to the holding pipet. The pronucleus that will be injected, the holding pipet, and the injection tip are all in focus. (B) Diagram of the same arrangement. Note the orientation of the zygote is such that the pronucleus that will be injected is adjacent to the injection pipet. (C) Diagram illustrating the injection tip after penetration. The pronucleus should swell visibly, but not lyse.
    View Image
  •  FigureFigure 23.9.7 Oviduct Reimplantation. (A) Photograph showing the location of the central skin incision site. Note that the area around the site has been shaved. (B) Photograph showing the orientation of the mouse for reimplantation with an extruded ovary and fat pad held by a serrefine clamp.
    View Image
  •  FigureFigure 23.9.8 Reimplantation in the oviduct. (A) Photograph of the oviduct ready for reimplantation. (B) Diagram of the oviduct and alternative reimplantation sites. Note the location of the puncture site for puncture transfers and the infundibulum for infundibular transfers.
    View Image
  •  FigureFigure 23.9.9 Injection apparatus. Photograph of a typical injection apparatus. This mixed system is based on a Zeiss Axiovert microscope with Narishige manipulators. The microinjector (not shown) for the holding pipet is a simple threaded syringe resting on the right side of the microscope connected by tubing to the pipet holder on the left side. The micromanipulator for the holding pipet is on the left side. Also on the left-hand side is an Eppendorf FemtoJet microinjector. This can be activated by a foot pedal (not shown). The entire system rests on an air isolation table.
    View Image

Videos

Literature Cited

Literature Cited
    Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Senear, A.W., Warren, R., and Palmiter, R.D. 1981. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27:223-231.
    Constantini, F. and Lacy, E. 1981. Introduction of a rabbit beta-globin gene into the mouse germ line. Nature 294:92-94.
    Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A, and Ruddle, FH. 1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. U.S.A. 77:7380-7384.
    Harbers, K., Jahner, D., and Jaenisch, R. 1981. Microinjection of cloned retroviral genomes into mouse zygotes: Integration and expression in the animal. Nature 293:540-542.
    Jahner, D., Haase, K., Mulligan, R., and Jaenisch, R. 1985. Insertion of the bacterial gpt gene into the germ line of mice by retroviral infection. Proc. Natl. Acad. Sci. U.S.A. 82:6927-6931.
    Lee, E.C., Yu, D., Martinez de Velasco, J., Tessarollo, L., Swing, D.A., Court, D.L., Jenkins, N.A., and Copeland, N.G. 2001. A highly efficient Escherichia coli–based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 72:56-65.
    Lois, C., Hong, E.J., Pease, S., Brown, E.J., and Baltimore, D. 2002. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295:868-872.
    Montoliu, L., Bock, C.-T., Schutz, G., and Zentgraf, H. 1995. Visualization of large DNA molecules by electron microscopy with polyamines: Application to the analysis of yeast endogenous and artificial chromosomes. J. Mol. Biol. 246:486-492.
    Schedl, A., Beermann, F., Thies, E., Montoliu, L., Kelsey, G., and Schutz, G. 1992. Transgenic mice generated by pronuclear injection of a yeast artificial chromosome. Nucl. Acids Res. 20:3073-3077.
    Strauss, W.M., Dausman, J., Beard, C., Johnson, C., Lawrence, J.B., and Jaenisch, R. 1993. Germ line transmission of a yeast artificial chromosome spanning the murine alpha 1(I) collagen locus. Science 259:1904-1907.
    Testa, G., Vintersten, K., Zhang, Y., Benes, V., Muyrers, J.P., and Stewart, A.F. 2004. BAC engineering for the generation of ES cell-targeting constructs and mouse transgenes. Methods Mol. Biol. 256:123-139.
    van der Putten, H., Botteri, F.M., Miller, A.D., Rosenfeld, M.G., Fan, H., Evans, R.M., and Verma, I.M. 1985. Efficient insertion of genes into the mouse germ line via retroviral vectors. Proc. Natl. Acad. Sci. U.S.A. 82:6148-6152.
    Wagner, E.F, Stewart, T.A., and Mintz, B. 1981a. The human beta-globin gene and a functional viral thymidine kinase gene in developing mice. Proc. Natl. Acad. Sci. U.S.A. 78:5016-5020.
    Wagner, T.E., Hoppe, P.C., Jollick, J.D., Scholl, D.R., Hodinka, R.L., and Gault, J.B. 1981b. Microinjection of a rabbit beta-globin gene in zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl. Acad. Sci. U.S.A. 78:6376-6380.
 Key References
    Nagy, A., Gertsenstein, M., Vinterstein, K., and Behringer, R. 2003. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

This manual is a compendium of methods used to modify the mouse genome, analyze genetically altered embryos, and maintain mouse lines. It is probably the most-referenced text on transgenic mouse production.

    Hammes, A. and Schedl, A. 2000. Mouse Genetics and Transgenics: A Practical Approach (I.J. Jackson and C.M. Abbott, eds.) pp. 217-245. Oxford University Press Inc., New York.
    Voncken, J.W. 2003. Transgenic Mouse: Methods and Protocols (M.H. Hofker and J. van Deursen, eds.) pp. 9-34. Humana Press, Totawa, New Jersey.

The last two references are also collections of methods used to modify the mouse genome. They overlap the first reference with regard to transgenic mouse production, but provide additional protocols for the genetic analysis of mice and further characterization of transgenic animals.

 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 transgenics by pronuclear injection.

    http://www.jax.org

The Jackson Laboratory web page provides links to a wealth of mouse related information, including, mutant resources, trait mapping resources, and literature pertaining to mouse genetics and animal husbandry.

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