Skip to main content
SpringerLink
Log in

Pronuclear Microinjection of Mouse Zygotes

  • Protocol
Germ Cell Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 254))

Abstract

The most important tools to investigate the genome of an organism are spontaneous or induced mutations. In mammals, with the exception of humans, the mouse is genetically the most thoroughly analyzed species. Hundreds of different mutant mouse strains are being bred in laboratories and stored as frozen embryos or germ cells in repositories around the world. Because spontaneous mutations are very rare events, different methods to increase the incidence of mutagenesis were developed several decades ago. One example is the alkylating agent N-ethyl-N-nitrosourea, a powerful tool for producing random point mutations in premeiotic spermatogonia. This “phenotype driven” approach is most helpful to identify unknown genes and their function. Complementary to this large-scale random mutagenesis technique are the so called “gene-driven” approaches, which involve selective manipulation of the mouse genome with the objective of creating transgenic mice. In contrast to chemical mutagenesis, where phenotypic changes are a prerequisite to identify the induced mutation (forward genetics), transgenic technology is based on the reintroduction of a previously isolated and in vitro recombined DNA sequence (reverse genetics).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gordon, J. W. and Ruddle, F. H. (1981) Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214, 1244–1246.

    Article  PubMed  CAS  Google Scholar 

  2. Rulicke, T. (1996) Transgenic technology: an introduction. Int. J. Exp. Pathol. 77, 243–245.

    PubMed  CAS  Google Scholar 

  3. Chan, A. W., Homan, E. J., Ballou, L. U., Burns, J. C., and Bremel, R. D. (1998) Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proc. Natl. Acad. Sci. USA 95, 14,028–14,0233.

    Article  PubMed  CAS  Google Scholar 

  4. Nagano, M., Brinster, C. J., Orwig, K. E., Ryu, B. Y., Avarbock, M. R., and Brinster, R. L. (2001) Transgenic mice produced by retroviral transduction of male germ-line stem cells. Proc. Natl. Acad. Sci. USA 98, 13,090–13,905.

    Article  PubMed  CAS  Google Scholar 

  5. Jaenisch, R. (1976) Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. USA 73, 1260–1264.

    Article  PubMed  CAS  Google Scholar 

  6. Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., and Ruddle, F. H. (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77, 7380–7384.

    Article  PubMed  CAS  Google Scholar 

  7. Mansour, S. L., Thomas, K. R., Deng, C. X., and Capecchi, M. R. (1990) Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination. Proc. Natl. Acad. Sci. USA 87, 7688–7692.

    Article  PubMed  CAS  Google Scholar 

  8. Lin, T. P. (1966) Microinjection of mouse eggs. Science 151, 333–337.

    Article  PubMed  CAS  Google Scholar 

  9. Taketo, M., Schroeder, A. C., Mobraaten, L. E., Gunning, K. B., Hanten, G., Fox, R. R., et al. (1991) FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc. Natl. Acad. Sci. USA 88, 2065–2069.

    Article  PubMed  CAS  Google Scholar 

  10. Mouse Genome Database (MGD), Mouse Genome Informatix Web Site. The Jackson Laboratory, Bar Harbor, Maine, http://www.informatics.jax.org.

  11. Whitten, W. K. (1956) Modifications of the oestrous cycle of the mouse by external stimuli associated with the male. J. Endocr. 13, 399–404.

    Article  PubMed  CAS  Google Scholar 

  12. Behrens, A., Genoud, N., Naumann, H., Rülicke, T., Janett, F., Heppner, F., et al. (2002) Absence of the prion protein homologue Doppel causes male sterility. EMBO J. 21, 1–7.

    Article  Google Scholar 

  13. Arras, M., Autenried, P., Rettich, A., Spaeni, D., and Rülicke, T. (2001) Optimization of intraperetonial injection anesthesia in mice: drugs, dosages adverse effects, and anesthesia depth. Comparative Medi. 51, 443–456.

    CAS  Google Scholar 

  14. Bruce, H. M. (1959) An exteroceptive block to pregnancy in the mouse. Nature Lond. 184, 109.

    Google Scholar 

  15. Wong, E. A. and Capecchi, M. R. (1985) Effect of cell cycle position on transformation by microinjection. Somat. Cell Mol. Genet. 11, 43–51.

    Article  PubMed  CAS  Google Scholar 

  16. Roth, D. B. and Wilson, J. H. (1985) Relative rates of homologous and nonhomologous recombination in transfected DNA. Proc. Natl. Acad. Sci. USA 82, 3355–3359.

    Article  PubMed  CAS  Google Scholar 

  17. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., and Palmiter, R. D. (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82, 4438–4442.

    Article  PubMed  CAS  Google Scholar 

  18. Koetsier, P. A., Mangel, L., Schmitz, B., and Doerfler, W. (1996) Stability of transgene methylation patterns in mice: position effects, strain specificity and cellular mosaicism. Transgenic Res. 5, 235–244.

    Article  PubMed  CAS  Google Scholar 

  19. Schedl, A., Larin, Z., Montoliu, L., Thies, E., Kelsey, G., Lehrach, H., and Schutz, G. (1993) A method for the generation of YAC transgenic mice by pronuclear microinjection. Nucleic Acids Res. 21, 4783–4787.

    Article  PubMed  CAS  Google Scholar 

  20. Mrkic, B., Pavlovic, J., Rulicke, T., Volpe, P., Buchholz, C. J., Hourcade, D., et al. (1998) Measles virus spread and pathogenesis in genetically modified mice. J. Virol. 72, 7420–7427.

    PubMed  CAS  Google Scholar 

  21. Zirkin, B. R., Perreault, S. D., and Naish, S. J. Formation and function of the paternal pronucleus during mammalian fertilization, in Molecular Biology of Fertilization (Schatten, H. S. and Schatten, G., ed.), Academic Press, San Diego, CA, 1989.

    Google Scholar 

  22. Rulicke, T. and Autenried, P. (1995) Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation. Lab Anim 29(3), 320–326.

    Article  PubMed  CAS  Google Scholar 

  23. van der Meer, M., Costa, P., Baumans, V., Olivier, B., and van Zutphen, B. (1999) Welfare assessment of transgenic animals: Behavioural responses and morphological development of newborn mice. ATLA 27, 857–868.

    Google Scholar 

  24. International Committee on Standardized Genetic Nomenclature for Mice (2000) Mouse Nomenclature Home Page. Rules and Guidelines for Gene, Allele, and Mutation Nomenclature. The Jackson Laboratory, Bar Harbor, Maine. http://www.informatics.jax.org.

    Google Scholar 

  25. Hogan, B., Beddington, R., Costantini, F., and Lacy, E. Manipulating the Mouse Embryo. A laboratory manual. 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1994.

    Google Scholar 

  26. Henneberger, C., Grantyn, R., and Rothe, T. (2000) Rapid genotyping of newborn gene mutant mice. J. Neurosci. Methods 100(1–2), 123–126.

    Article  PubMed  CAS  Google Scholar 

  27. Ren, S., Li, M., Cai, H., Hudgins, S., and Furth, P. A. (2001) A simplified method to prepare PCR template DNA for screening of transgenic and knockout mice. Contemp. Top. Lab. Anim. Sci. 40, 27–30.

    PubMed  CAS  Google Scholar 

  28. Malumbres, M., Mangues, R., Ferrer, N., Lu, S., and Pellicer, A. (1997) Isolation of high molecular weight DNA for reliable genotyping of transgenic mice. Biotechniques 22, 1114–9.

    PubMed  CAS  Google Scholar 

  29. Campbell, D. B. and Hess, E. J. (1997) Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis. Brain Res. Brain Res. Protoc. 1, 117–123.

    Article  PubMed  CAS  Google Scholar 

  30. Broome, R. L., Feng, L., Zhou, Q., Smith, A., Hahn, N., Matsui, S. M., and Omary, M. B. (1999) Non-invasive transgenic mouse genotyping using stool analysis. FEBS Lett. 462(1–2), 159–160.

    Article  PubMed  CAS  Google Scholar 

  31. Lahm, H., Hoeflich, A., Rieger, N., Wanke, R., and Wolf, E. (1998) Identification of transgenic mice by direct PCR analysis of lysates of epithelial cells obtained from the inner surface of the rectum. Transgenic Res. 7, 131–134.

    Article  PubMed  CAS  Google Scholar 

  32. Zimmermann, K., Schwarz, H. P., and Turecek, P. L. (2000) Deoxyribonucleic acid preparation in polymerase chain reaction genotyping of transgenic mice. Comp. Med. 50, 314–316.

    PubMed  CAS  Google Scholar 

  33. Irwin, M. H., Moffatt, R. J., and Pinkert, C. A. (1996) Identification of transgenic mice by PCR analysis of saliva. Nat. Biotechnol. 14, 1146–1148.

    Article  PubMed  CAS  Google Scholar 

  34. Schmitteckert, E. M., Prokop, C. M., and Hedrich, H. J. (1999) DNA detection in hair of transgenic mice114-a simple technique minimizing the distress on the animals. Lab. Anim. 33, 385–389.

    Article  PubMed  CAS  Google Scholar 

  35. Cousens, C., Carver, A. S., Wilmut, I., Colman, A., Garner, I., and O’Neill, G. T. (1994) Use of PCR-based methods for selection of integrated transgenes in preimplantation embryos. Mol. Reprod. Dev. 39, 384–391.

    Article  PubMed  CAS  Google Scholar 

  36. Bishop, J. O. and Smith, P. (1989) Mechanism of chromosomal integration of microinjected DNA. Mol. Biol. Med. 6, 283–298.

    PubMed  CAS  Google Scholar 

  37. Aigner, B. and Brem, G. (1995) Detection of homozygous individuals in gene transfer experiments by semiquantitative PCR. Biotechniques 18, 754–756, 758.

    PubMed  CAS  Google Scholar 

  38. Linder, C. C. (2001) The influence of genetic background on spontaneous and genetically engineered mouse models of complex diseases. Lab. Anim. 30, 34–39.

    CAS  Google Scholar 

  39. Gerlai, R. (1996) Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci. 19, 177–181.

    Article  PubMed  CAS  Google Scholar 

  40. Wolfer, D. P., Muller, U., Stagliar, M., and Lipp, H. P. (1997) Assessing the effects of the 129/Sv genetic background on swimming navigation learning in transgenic mutants: a study using mice with a modified beta-amyloid precursor protein gene. Brain Res. 771, 1–13.

    Article  PubMed  CAS  Google Scholar 

  41. Nadeau, J. H. (2001) Modifier genes in mice and humans. Nat. Rev. Genet. 2, 165–174.

    Article  PubMed  CAS  Google Scholar 

  42. Festing, M. F. W. Inbred Strains in Biomedical Research. Medical Research Council Laboratory Animals Centre Carshalton, Surrey, UK, The Macmillan Press Ltd., London and Basingsoke, 1979.

    Google Scholar 

  43. Markel, P., Shu, P., Ebeling, C., Carlson, G. A., Nagle, D. L., Smutko, J. S., and Moore, K. J. (1997) Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains. Nat. Genet. 17, 280–284.

    Article  PubMed  CAS  Google Scholar 

  44. Behringer, R. (1998) Supersonic congenics? Nat. Genet. 18, 108.

    Article  PubMed  CAS  Google Scholar 

  45. Mutant mice and neuroscience: recommendations concerning genetic background. Banbury Conference on genetic background in mice. (1997) Neuron 19, 755–759.

    Google Scholar 

  46. Ward, J. M., Mahler, J. F., Maronpot, R. R., Sundberg, J. P., Frederickson, R. M. Pathology of genetically engineered mice. 1st edition, Iowa State University Press, Ames, IA, 2000.

    Google Scholar 

  47. Emerman, M. and Temin, H. M. (1984) Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39(3 Pt 2), 449–467.

    PubMed  CAS  Google Scholar 

  48. Bahramian, M. B. and Zarbl, H. (1999) Transcriptional and posttranscriptional silencing of rodent alpha1(I) collagen by a homologous transcriptionally self-silenced transgene. Mol. Cell Biol. 19, 274–283.

    PubMed  CAS  Google Scholar 

  49. Mertens, C. and Rulicke, T. (1999) Score sheets for the monitoring of transgenic mice. Animal Welfare 8, 433–438.

    Google Scholar 

  50. Mertens, C. and Rulicke, T. (2000) Phenotype characterization and welfare assessment of transgenic rodents (mice) J. Appl. Animal Welfare Sci. 3, 127–139.

    Article  Google Scholar 

  51. Crabbe, J. C., Wahlsten, D., and Dudek, B. C. (1999) Genetics of mouse behavior: interactions with laboratory environment. Science 284, 1670–1672.

    Article  PubMed  CAS  Google Scholar 

  52. Rampon, C., Tang, Y. P., Goodhouse, J., Shimizu, E., Kyin, M., and Tsien, J. Z. (2000) Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice. Nat. Neurosci. 3, 238–244.

    Article  PubMed  CAS  Google Scholar 

  53. Rulicke, T. and Hubscher, U. (2000) Germ line transformation of mammals by pronuclear microinjection. Exp. Physiol. 85, 589–601.

    Article  PubMed  CAS  Google Scholar 

  54. Garrick, D., Fiering, S., Martin, D. I., and Whitelaw, E. (1998) Repeat-induced gene silencing in mammals. Nat. Genet. 18, 56–59.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Laboratory Animal Science, University of Zurich, Zurich, Switzerland

    Thomas Rülicke

Authors
  1. Thomas Rülicke
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO

    Heide Schatten

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc.

About this protocol

Cite this protocol

Rülicke, T. (2004). Pronuclear Microinjection of Mouse Zygotes. In: Schatten, H. (eds) Germ Cell Protocols. Methods in Molecular Biology™, vol 254. Humana Press. https://doi.org/10.1385/1-59259-741-6:165

Download citation

  • DOI: https://doi.org/10.1385/1-59259-741-6:165

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-257-5

  • Online ISBN: 978-1-59259-741-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation