Advertisement

Cre Recombinase Mediated Alterations of the Mouse Genome Using Embryonic Stem Cells

  • Anna-Katerina Hadjantonakis
  • Melinda Pirity
  • András Nagy
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 461)

1. Introduction

The introduction and establishment of transgenic, and in particular embryonic stem (ES) cell-based gene “knockout” technologies have made the mouse a key player in studying embryonic development and disease (1,2). In recent years, methods for the production of more complex genomic alterations have become increasingly widespread, hinting at an ability to manipulate and study a mammalian genome to an extent never previously thought possible. Such methodologies often partner homologous recombination-mediated gene targeting or random integration with site-specific recombination events.

This chapter is concerned with the utilization of the bacteriophage P1 derived site-specific recombinase protein Cre (3, 4, 5), and its employment as a means to catalyze modifications in homologously recombined and randomly integrated target sites within the mouse genome.

Cre is a 38-kDa protein that recombines DNA between two loxP target sites. loxP sequences are 34 basepairs (bp) long...

Keywords

Embryonic Stem Embryonic Stem Cell Embryonic Stem Cell Line loxP Site Embryonic Stem Cell Culture 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Capecchi, M. R. (1989) The new mouse genetics: altering the genome by gene targeting. Trends Genet. 5, 70–76.CrossRefPubMedGoogle Scholar
  2. 2.
    Thomas, K. R., Deng, C., and Capecchi, M. R. (1992) High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol. Cell. Biol. 12, 2919–2923.PubMedGoogle Scholar
  3. 3.
    Sternberg, N. (1994) The P1 cloning system: past and future. Mamm. Genome 5,397–404.CrossRefPubMedGoogle Scholar
  4. 4.
    Argos, P., Landy, A., Abremsky, K., Egan, J. B., Haggard-Ljungquist, E., Hoess, R. H., et al. (1986) The integrase family of site-specific recombinases: regional similarities and global diversity. EMBO J. 5, 433–440.PubMedGoogle Scholar
  5. 5.
    Sauer, B. and Henderson, N. (1988) Site-specific DNA recombination in mamalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. USA 85,5166–5170.CrossRefPubMedGoogle Scholar
  6. 6.
    Hoess, R. H. snd Abremski, K. (1985) Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J. Mol. Biol. 181, 351–362.CrossRefPubMedGoogle Scholar
  7. 7.
    Babineau, D., Vetter, D., Andrews, B. J., Gronostajski, R. M., Proteau, G. A., Beatty, L. G., et al. (1985) The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene.J. Biol. Chem. 260, 12,313–12,319.Google Scholar
  8. 8.
    Cox, M. M. (1988) in Genetic Recombination (Kucherlapati, R. and Smith, G. R., eds.), American Society for Microbiology, Washington DC, pp. 429–443.Google Scholar
  9. 9.
    O'Gorman, S., Fox, D. T., and Wahl, G. M. (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251, 1351–1355.CrossRefPubMedGoogle Scholar
  10. 10.
    Fiering, S., Kim, C. G., Epner, E. M., and Groudine, M. (1993) An “in-out”strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the b-globin locus control region.Proc. Natl. Acad. Sci. USA 90, 8469–8473.CrossRefPubMedGoogle Scholar
  11. 11.
    Buchholz, F., Ringrose, L., Angrand, P.O., Rossi, F. and Stewart, A.F. (1996) Different thrermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucleic Acids Res. 24, 4256–4262.CrossRefPubMedGoogle Scholar
  12. 12.
    Rossant, J. and Nagy, A. (1995) Genome engineering: the new mouse genetics.Nat. Med. 1, 592–594.CrossRefPubMedGoogle Scholar
  13. 13.
    Nagy, A. (1996) in Mammalian Development (Lonai, P., ed.), Harwood Academic Publishers, Amsterdam, pp. 339–371.Google Scholar
  14. 13a.
    13a. Nagy, A., Moens, C., Ivanyi, E., Pawling, J. Gertsenstein, M,. Hadjantonakis, K., Pirity, M., and Rossant, J. (1998) Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles. Curr. Biol. 82,661–664.CrossRefGoogle Scholar
  15. 14.
    Gu, H., Zou, Y. R., and Rajewsky, K. (1993) Independent control of immunoglobu-lin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73, 1155–1164.CrossRefPubMedGoogle Scholar
  16. 15.
    Smith, A. J. H., Sousa, M. A. D., Kwabi-Addo, B., Heppell-Parton, A., Impey, H., and Rabbitts, P. (1995) A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nature Genetics 9, 376–385.CrossRefPubMedGoogle Scholar
  17. 16.
    Ramirez-Solis, R., Liu, P., and Bradley, A. (1995) Chromosome engineering in mice. Nature 378, 720–724.CrossRefPubMedGoogle Scholar
  18. 17.
    Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, J. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424–8428.CrossRefPubMedGoogle Scholar
  19. 18.
    Nagy, A. and Rossant, J. (1996) Targeted mutagenesis: Analysis of phenotype without germ line transmission. J. Clin. Invest. 97, 1360–1365.CrossRefPubMedGoogle Scholar
  20. 19.
    Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., et al. (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435–439.CrossRefPubMedGoogle Scholar
  21. 20.
    Zhang, H. & Bradley, A. (1996) Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 122, 2977–2986.PubMedGoogle Scholar
  22. 21.
    Takahama, Y., Ohishi, K., Tokoro, K., Sugawara, T., Yoshimura, Y., Okabe, M., et al.(1997) T cell-specific disruption of Pig-a gene involved in glycosylphosphatidyli-nositol (GPI) biosynthesis: Generation and function of T cells deficient in GPI-anchored proteins. Proc. Natl. Acad. Sci. USA, in press.Google Scholar
  23. 22.
    Tsien, J. Z., Chen, D. F., Gerber, D., Tom, C., Mercer, E. H., Anderson, D. J., et al.(1996) Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87,1317–1326.CrossRefPubMedGoogle Scholar
  24. 23.
    Bonner, J. J., Heyward, S., and Fackenthal, D. L. (1992) Temperature-dependent regulation of heterologous transcriptional activation domain fused to yeast heatshock transcription factor. Mol. Cell. Biol. 12, 1021–1030.PubMedGoogle Scholar
  25. 24.
    Palmiter, R. D., Norstedt, G., Gelinas, R. E., Hammer, R. E., and Brinster, R. L.(1983) Metallothionein-human GH fusion genes stimulate growth of mice. Science 222, 809–814.CrossRefPubMedGoogle Scholar
  26. 25.
    No, D., Yao, T. P., and Evans, R. M. (1996) Ecdysone-inducible gene expression in mammalian cells and transgenic mouse. Proc. Natl. Acad. Sci. USA 93, 3346–3351.CrossRefPubMedGoogle Scholar
  27. 26.
    Christopherson, K. S., Mark, M. R., Bajaj, V., and Godowski, P. J. (1992) Ecdyster-oid-dependent regulation of genes in mammalian cells by a Drosophila ecdysone receptor and chimeric transactivators. Proc. Natl. Acad. Sci. USA 89, 6314–6318.CrossRefPubMedGoogle Scholar
  28. 27.
    Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cell by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551.CrossRefPubMedGoogle Scholar
  29. 28.
    Rivera, V. M., Clackson, T., Natesan, S., Pollock, R., Amara, J., Keenan, T., et al.(1996) A humanized system for pharmacologic control of gene expression. Nat.Med. 2, 1028–1032.CrossRefPubMedGoogle Scholar
  30. 29.
    Lawson, K. A. and Pedersen, R. A. (1987) Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse. Development 101, 627–652.PubMedGoogle Scholar
  31. 30.
    Smith, J. L., Gesteland, K. M., and Schoenwolf, G. C. (1994) Prospective fate map of the mouse primitive streak at 7.5 days of gestation. Dev. Dyn. 201, 279–289.PubMedGoogle Scholar
  32. 31.
    Gossler, A., Joyner, A. L., Rossant, J., and Skarnes, W. C. (1989) Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.Science 244, 463–465.CrossRefPubMedGoogle Scholar
  33. 32.
    Joyner, A. L., Auerbach, A., and Scarnes.W.C. (1992) The gene trap approach in embryonic stem cells: the potential for genetic screens in mice. Ciba Foundation Symp.165, 277–288.Google Scholar
  34. 33.
    Forrester, L. M., Nagy, A., Sam, M., Watt, A., Stevenson, L., Bernstein, A., et al.(1996) An induction gene trap screen in embryonic stem cells: Identification of genes that respond to retinoic acid in vitro. Proc. Natl. Acad. Sci. USA 93, 1677–1682.CrossRefGoogle Scholar
  35. 34.
    Sauer, B. and Henderson, N. (1989) Cre-stimulated recombination at loxP-containing DNA equences placed into the mammalian genome. Nucleic Acids Res. 17,147–161.CrossRefPubMedGoogle Scholar
  36. 35.
    Sauer, B. and Henderson, N. (1990) Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase. New Biologist 2, 441–449.PubMedGoogle Scholar
  37. 36.
    Sauer, B. (1993) in Guide to Techniques in Mouse Development, Methods in Embryology, vol 225 (Wasserman, P. M. and DePamphilis, M. L., eds.), Academic, San Diego, pp. 890–900.CrossRefGoogle Scholar
  38. 37.
    Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morph. 87,27–45.PubMedGoogle Scholar
  39. 38.
    Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D.P., et al. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687.CrossRefPubMedGoogle Scholar
  40. 39.
    Wurst, W. and Joyner, A. L. (1993) Gene Targeting: A Practical Approach. (Joyner, A. L., ed.), Oxford University Press, Oxford, p. 33.Google Scholar
  41. 40.
    Nagy, A., Merentes, E., Gocza, E., Ivanyi, E., and Rossant, J. (1991) Develop-metal potential of mouse embryonic stem cells and inner cell mass: a comparison.[Abstract] Symposium of Mouse Molecular Genetics, Heidelberg, August 21–25,1991.Google Scholar
  42. 41.
    Schwenk, F., Baron, U., and Rajewsky, K. (1995) A cre-transgenic mouse strain for the ubiquitous deletion ofloxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 23, 5080,5081.CrossRefPubMedGoogle Scholar
  43. 42.
    Araki, K., Araki, M., Miyazaki, J., and Vassalli, P. (1995) Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase.Proc. Natl. Acad. Sci. USA 92, 160–164.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Anna-Katerina Hadjantonakis
    • 1
  • Melinda Pirity
    • 2
  • András Nagy
    • 2
  1. 1.Memorial Sloan Kettering Cancer CenterNew YorkUSA
  2. 2.Samuel Lunenfeld Research InstituteMount Sinai HospitalTorontoCanada

Personalised recommendations