Molecular Biotechnology

, Volume 19, Issue 3, pp 297–304 | Cite as

Contemporary gene targeting strategies for the novice

Protocol

Abstract

Gene targeting in mouse embryonic stem (ES) cells is a fundamental methodology for generating mice with precise genetic modifications. Although there are many complex gene targeting strategies for creating a variety of diverse mutations in mice, most investigators initially choose to generate a null allele. Here we provide a guide for the novice to generate a null allele for a protein coding gene using a fundamental gene targeting strategy. Ultimately, a well considered gene targeting strategy saves significant amounts of time, money, and research animal lives. The straightforward strategy presented here bypasses many of the pitfalls associated with gene knockouts generated by novices. This guide also serves as a foundation for subsequently designing more complex gene targeting strategies.

Index Entries

Gene targeting embryonic stem cells Cre/loxP knockout homologous recombination 

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References

  1. 1.
    Torres, M. (1998) The use of embryonic stem cells for the genetic manipulation of the mouse. Curr. Top. Dev. Biol. 36, 99–114.PubMedCrossRefGoogle Scholar
  2. 2.
    Bradley, A. and Liu, P. (1996) Target practice in transgenics. Nature Gen. 14, 121–123.CrossRefGoogle Scholar
  3. 3.
    Chen, Z.-F. and Behringer, R. R. (1995) twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev. 9, 686–699.PubMedGoogle Scholar
  4. 4.
    Rivera-Pérez, J. A., Mallo, M., Gendron-Maguire, M., et al. (1995) Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development. Development 121, 3005–3012.PubMedGoogle Scholar
  5. 5.
    Shawlot, W. and Behringer, R. R. (1995) Requirement for Lim1 in head-organizer function. Nature 374, 425–430.PubMedCrossRefGoogle Scholar
  6. 6.
    Moens, C. B., Auerbach, A. B., Conlon, R. A., et al. (1992) Targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Dev. 6, 691–704.PubMedGoogle Scholar
  7. 7.
    Hasty, P., Bradley, A., Morris, J. H., et al. (1993) Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364, 501–506.PubMedCrossRefGoogle Scholar
  8. 8.
    Mombaerts, P., Clarke, A. R., Hooper, M. L., et al. (1991) Creation of a large genomic deletion at the T-cell antigen receptor beta-subunit locus in mouse embryonic stem cells by gene targeting. Proc. Natl. Acad. Sci. USA 88, 3084–3087.PubMedCrossRefGoogle Scholar
  9. 9.
    Zhang, H., Hasty, P., and Bradley, A. (1994) Targeting frequency for deletion vectors in embryonic stem cells. Mol. Cell. Biol. 14, 2404–2410.PubMedGoogle Scholar
  10. 10.
    Tsuda, H., Maynard-Currie, C. E., Reid, L. H., et al. (1997) Inactivation of the mouse HPRT locus by a 203-bp retroposon insertion and a 55-kb gene-targeted deletion: establishment of new HPRT-deficient mouse embryonic stem cell lines. Genomics 15, 413–421.CrossRefGoogle Scholar
  11. 11.
    Ramírez-Solis, R., Liu, P., and Bradley, A. (1995) Chromosome engineering in mice. Nature 378, 720–724.PubMedCrossRefGoogle Scholar
  12. 12.
    Li, Z. W., Stark, G., Gotz, J., et al. (1996) Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells. Proc. Natl. Acad. Sci. USA 93, 6158–6162.PubMedCrossRefGoogle Scholar
  13. 13.
    Mishina, Y., Suzuki, A., Ueno, N., et al. (1995) Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9, 3027–3037.PubMedGoogle Scholar
  14. 14.
    Mishina, Y., Rey, R., Finegold, M. J., et al. (1996) Genetic analysis of the Mullerian-inhibiting substance signal transduction pathway in mammalian sexual differentiation. Genes Dev. 10, 2577–2587.PubMedGoogle Scholar
  15. 15.
    Hasty, P. and Bradley, A. (1993) Gene targeting vectors for mammalian cells, in Gene Targeting: A Practical Approach (Joyner, A. L., ed.) IRL Press, Oxford, pp. 1–31.Google Scholar
  16. 16.
    Mansour, S. L., Thomas, K. R., and Capecchi, M. R. (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352.PubMedCrossRefGoogle Scholar
  17. 17.
    Ramírez-Solis, R., Rivera-Pérez, J., Wallace, J. D., et al. (1992) Genomic DNA microextraction: a method to screen numerous samples. Analyt. Biochem. 201, 331–335.PubMedCrossRefGoogle Scholar
  18. 18.
    Wood, S. A., Allen, N. D., Rossant, J., et al. (1993) Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365, 87–89.PubMedCrossRefGoogle Scholar
  19. 19.
    Robertson, E. J. (1987) Embryo-derived stem cell lines, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (Robertson, E. J., ed.) IRL Press, Oxford, pp. 71–112.Google Scholar
  20. 20.
    Holdener, B. C., Faust, C., Rosenthal, N. S., et al. (1994) msd is required for mesoderm induction in mice. Development 120, 1335–1346.PubMedGoogle Scholar
  21. 21.
    te Riele, H., Maandag, E. R., and Berns, A. (1992) Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. USA 89, 5128–5132.CrossRefGoogle Scholar
  22. 22.
    Papaioannou, V. and Johnson, R. (1993) Production of chimeras and genetically defined offspring from targeted ES cells, in Gene Targeting: A Practical Approach (Joyner, A. L., ed.) IRL Press, Oxford, pp. 1–31.Google Scholar
  23. 23.
    Threadgill, D. W., Yee, D., Matin, A., et al. (1997) Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain. Mamm. Genome 8, 390–393.PubMedCrossRefGoogle Scholar
  24. 24.
    Nagy, A., Rossant, J., Nagy, R., et al. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424–8428.PubMedCrossRefGoogle Scholar
  25. 25.
    Dresser, D. W., Hacker, A., Lovell-Badge, R., et al. (1995) The genes for a spliceosome protein (SAP62) and the anti-Mullerian hormone (AMH) are contiguous. Hum. Mol. Genet. 4, 1613–1618.PubMedCrossRefGoogle Scholar
  26. 26.
    Soriano, P., Montgomery, C., Geske, R. and Bradley, A. (1991) Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64, 693–702.PubMedCrossRefGoogle Scholar
  27. 27.
    Fiering, S., Epner, E., Robinson, K., et al. (1995) Targeted deletion of 5′HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. Genes Dev. 9, 2203–2213.PubMedGoogle Scholar
  28. 28.
    Nagy, A. (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109.PubMedCrossRefGoogle Scholar
  29. 29.
    Olson, E. N., Arnold, H. H., Rigby, P. W., et al. (1996) Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 85, 1–4.PubMedCrossRefGoogle Scholar
  30. 30.
    Kaul, A., Köster, M., Neuhaus, H., and Braun, T. (2000) Myf-5 Revisited: Loss of Early Myotome Formation Does Not Lead to a Rib Phenotype in Homozygous Myf-5 Mutant Mice. Cell 102, 17–19.PubMedCrossRefGoogle Scholar
  31. 31.
    Abuin, A. and Bradley, A. (1996) Recycling selectable markers in mouse embryonic stem cells. Mol. Cell. Biol. 16, 1851–1856.PubMedGoogle Scholar
  32. 32.
    Quinn, J. C., West, J. D., and Hill, R. E. (1996) Multiple functions for Pax6 in mouse eye and nasal development. Genes Dev. 10, 435–446.PubMedGoogle Scholar
  33. 33.
    Rivera-Pérez, J. A., Wakamiya, M., and Behringer, R. R. (1999) Goosecoid acts cell autonomously for the maintenance of mesenchymal tissues during craniofacial development. Development. 126, 3811–3821.PubMedGoogle Scholar
  34. 34.
    Sunaga, S., Maki, K., Komagata, Y., et al. (1997) Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs. Mol. Reprod. Dev. 46, 109–113.PubMedCrossRefGoogle Scholar
  35. 35.
    Lasko, M., Sauer, B., Mosinger, B. Jr., et al. (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 6232–6236.CrossRefGoogle Scholar
  36. 36.
    McMahon, A. P. and Bradley, A. (1990) The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62, 1073–1085.PubMedCrossRefGoogle Scholar
  37. 37.
    McCarrick, J. W., Parnes, J. R., Seong, R. H., et al. (1993) Positive-negative selection gene targeting with the diphtheria toxin A-chain gene in mouse embryonic stem cells. Transgenic Res. 2, 183–190.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2001

Authors and Affiliations

  1. 1.Department of Molecular GeneticsThe University of Texas M. D. Anderson Cancer CenterHouston

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