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

  • 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, ⅜‐in. tuberculin syringes with 26‐G needles
  • Surgical equipment (see protocol 6):
    • 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 protocol 6)
  • 37°C, 5% CO 2 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 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 protocol 6)
  • Embryo transfer pipet (see protocol 6)
  • 37°C, 5% CO 2 incubator
  • Transgene in injection buffer (see Support Protocols protocol 41 or protocol 52)

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 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 ( protocol 6)
  • 1‐ml, ⅜‐in. tuberculin syringes with 26‐G needles
  • Surgical equipment and supplies ( protocol 6):
    • 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 protocol 6)
  • 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 2.5& 2.6), and ethanol precipitation (unit 2.1)

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 2.5)
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

Videos

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.
  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.
   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.
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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