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Recombinases and Their Use in Gene Activation, Gene Inactivation, and Transgenesis

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Drosophila

Part of the book series: Methods in Molecular Biology ((MIMB,volume 420))

Abstract

The site-specific recombinase FLP is used in Drosophila to precisely manipulate the genome, in particular, to eliminate gene function by mitotic recombination and to activate transgenes in discrete populations of cells. These approaches are already part of the standard tool kit for studying gene function. The number of applications for the FLP recombinase has increased over the years and further members of the large family of site-specific recombinases are being added to the arsenal of fly geneticists, most recently, the φC31 integrase. This chapter will introduce these recombinases and describe how such instruments are utilized to accurately manipulate the Drosophila genome.

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References

  1. Rubin, G. M. and Spradling, A. C. (1982) Genetic transformation of Drosophila with transposable element vectors. Science 218, 348–353.

    Article  CAS  PubMed  Google Scholar 

  2. Spradling, A. C. and Rubin, G. M. (1982) Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341–347.

    Article  CAS  PubMed  Google Scholar 

  3. Brand, A. H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.

    CAS  PubMed  Google Scholar 

  4. Duffy, J. B. (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34, 1–15.

    Article  CAS  PubMed  Google Scholar 

  5. Golic, K. G. and Lindquist, S. (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59, 499–509.

    Article  CAS  PubMed  Google Scholar 

  6. Kolb, A. F. (2002) Genome engineering using site-specific recombinases. Cloning Stem Cells 4, 65–80.

    Article  CAS  PubMed  Google Scholar 

  7. Stark, W. M., Boocock, M. R., and Sherratt, D. J. (1992) Catalysis by site-specific recombinases. Trends Genet. 8, 432–439.

    Article  CAS  PubMed  Google Scholar 

  8. Nagy, A. (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109.

    Article  CAS  PubMed  Google Scholar 

  9. Sorrell, D. A. and Kolb, A. F. (2005) Targeted modification of mammalian genomes. Biotechnol. Adv. 23, 431–469.

    Article  CAS  PubMed  Google Scholar 

  10. Nakano, M., Odaka, K., Ishimura, M., et al. (2001) Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus. Nucleic Acids Res. 29, E40.

    Article  CAS  PubMed  Google Scholar 

  11. Siegal, M. L. and Hartl, D. L. (1996) Transgene Coplacement and high efficiency site-specific recombination with the Cre/loxP system in Drosophila. Genetics 144, 715–726.

    CAS  PubMed  Google Scholar 

  12. Heidmann, D. and Lehner, C. F. (2001) Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation. Dev. Genes Evol. 211, 458–465.

    Article  CAS  PubMed  Google Scholar 

  13. Blair, S. S. (2003) Genetic mosaic techniques for studying Drosophila development. Development 130, 5065–5072.

    Article  CAS  PubMed  Google Scholar 

  14. Ashburner, M., Golic, K. G., and Hawley, R. S. (eds.) (2005) Drosophila. A laboratory handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  15. Struhl, G. and Basler, K. (1993) Organizing activity of wingless protein in Drosophila. Cell 72, 527–540.

    Article  CAS  PubMed  Google Scholar 

  16. de Celis, J. F. and Bray, S. (1997) Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124, 3241–3251.

    PubMed  Google Scholar 

  17. Ito, K.,Awano,W., Suzuki, K., Hiromi, Y., and Yamamoto, D. (1997) The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124, 761–771.

    CAS  PubMed  Google Scholar 

  18. Pignoni, F. and Zipursky, S. L. (1997) Induction of Drosophila eye development by decapentaplegic. Development 124, 271–278.

    CAS  PubMed  Google Scholar 

  19. Duffy, J. B., Harrison, D. A., and Perrimon, N. (1998) Identifying loci required for follicular patterning using directed mosaics. Development 125, 2263–2271.

    CAS  PubMed  Google Scholar 

  20. Weigmann, K. and Cohen, S. M. (1999) Lineage-tracing cells born in different domains along the PD axis of the developing Drosophila leg. Development 126, 3823–3830.

    CAS  PubMed  Google Scholar 

  21. Stern, C. (1934) Crossing over and segregation in somatic cells of Drosophila melanogaster. (Abstr.) Am. Nat. 68, 164–165.

    Google Scholar 

  22. Stern, C. (1936) Somatic crossing over and segregation in Drosophila melanogaster. Genetics 21, 625–730.

    CAS  PubMed  Google Scholar 

  23. Golic, K. G. (1991) Site-specific recombination between homologous chromosomes in Drosophila. Science 252, 958–961.

    Article  CAS  PubMed  Google Scholar 

  24. Chou, T. B. and Perrimon, N. (1992) Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics 131, 643–653.

    CAS  PubMed  Google Scholar 

  25. Xu, T. and Rubin, G. M. (1993) Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237.

    CAS  PubMed  Google Scholar 

  26. Chou, T. B., Noll, E., and Perrimon, N. (1993) Autosomal P[ovoD1] dominant female-sterile insertions in Drosophila and their use in generating germ-line chimeras. Development 119, 1359–1369.

    CAS  PubMed  Google Scholar 

  27. Chou, T. B. and Perrimon, N. (1996) The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144, 1673–1679.

    CAS  PubMed  Google Scholar 

  28. Lee, T. and Luo, L. (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461.

    Article  CAS  PubMed  Google Scholar 

  29. Golic, K. G. and Golic, M. M. (1996) Engineering the Drosophila genome: chromosome rearrangements by design. Genetics 144, 1693–1711.

    CAS  PubMed  Google Scholar 

  30. Parks, A. L., Cook, K. R., Belvin, M., et al. (2004) Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat. Genet. 36, 288–292.

    Article  CAS  PubMed  Google Scholar 

  31. Ryder, E., Blows, F., Ashburner, M., et al. (2004) The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster. Genetics 167, 797–813.

    Article  CAS  PubMed  Google Scholar 

  32. Baer, A. and Bode, J. (2001) Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes. Curr. Opin. Biotechnol. 12, 473–480.

    Article  CAS  PubMed  Google Scholar 

  33. Oberstein, A., Pare, A., Kaplan, L., and Small, S. (2005) Site-specific transgenesis by Cre-mediated recombination in Drosophila. Nat. Methods 2, 583–585.

    Article  CAS  PubMed  Google Scholar 

  34. Horn, C. and Handler, A. M. (2005) Site-specific genomic targeting in Drosophila. Proc. Natl. Acad. Sci. USA 102, 12,483–12,488.

    Article  CAS  PubMed  Google Scholar 

  35. Kuhstoss, S. and Rao, R. N. (1991) Analysis of the integration function of the streptomycete bacteriophage phi C31. J. Mol. Biol. 222, 897–908.

    Article  CAS  PubMed  Google Scholar 

  36. Rausch, H. and Lehmann, M. (1991) Structural analysis of the actinophage phi C31 attachment site. Nucleic Acids Res. 19, 5187–5189.

    Article  CAS  PubMed  Google Scholar 

  37. Thorpe, H. M., Wilson, S. E., and Smith, M. C. (2000) Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31. Mol. Microbiol. 38, 232–241.

    Article  CAS  PubMed  Google Scholar 

  38. Groth, A. C., Fish, M., Nusse, R., and Calos, M. P. (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166, 1775–1782.

    Article  CAS  PubMed  Google Scholar 

  39. Bateman, J. R., Lee, A. M., and Wu, C. T. (2006) Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange. Genetics 173, 769–777.

    Article  CAS  PubMed  Google Scholar 

  40. Venken, K. J., He, Y., Hoskins, R. A., and Bellen, H. J. (2006) P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogaster. Science 314, 1747–1751.

    Article  CAS  PubMed  Google Scholar 

  41. Venken, K. J. and Bellen, H. J. (2005) Emerging technologies for gene manipulation in Drosophila melanogaster. Nat. Rev. Genet. 6, 167–178.

    Article  CAS  PubMed  Google Scholar 

  42. Bischof, J., Maeda, R. K., Hediger, M., Karch, F., and Basler, K. (2007) An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases. Proc. Natl. Acad. Sci. USA 104, 3312–3317.

    Article  CAS  PubMed  Google Scholar 

  43. Sadowski, P. D. (1995) The Flp recombinase of the 2-microns plasmid of Saccharomyces cerevisiae. Prog. Nucleic Acid Res. Mol. Biol. 51, 53–91.

    Article  CAS  PubMed  Google Scholar 

  44. McLeod, M., Craft, S., and Broach, J. R. (1986) Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle. Mol. Cell Biol. 6, 3357–3367.

    CAS  PubMed  Google Scholar 

  45. Senecoff, J. F., Bruckner, R. C., and Cox, M. M. (1985) The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site. Proc. Natl. Acad. Sci. USA 82, 7270–7274.

    Article  CAS  PubMed  Google Scholar 

  46. Amin, A., Roca, H., Luetke, K., and Sadowski, P. D. (1991) Synapsis, strand scission, and strand exchange induced by the FLP recombinase: analysis with half-FRT sites. Mol. Cell Biol. 11, 4497–4508.

    CAS  PubMed  Google Scholar 

  47. Hoess, R. H., Ziese, M., and Sternberg, N. (1982) P1 site-specific recombination: nucleotide sequence of the recombining sites. Proc. Natl. Acad. Sci. USA 79, 3398–3402.

    Article  CAS  PubMed  Google Scholar 

  48. Branda, C. S. and Dymecki, S. M. (2004) Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28.

    Article  CAS  PubMed  Google Scholar 

  49. St Johnston, D. (2002) The art and design of genetic screens: Drosophila melanogaster. Nat. Rev. Genet. 3, 176–188.

    Article  CAS  PubMed  Google Scholar 

  50. Smith, M. C., Burns, R. N., Wilson, S. E., and Gregory, M. A. (1999) The complete genome sequence of the Streptomyces temperate phage straight phiC31: evolutionary relationships to other viruses. Nucleic Acids Res. 27, 2145–2155.

    Article  CAS  PubMed  Google Scholar 

  51. Thorpe, H. M. and Smith, M. C. (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc. Natl. Acad. Sci. USA 95, 5505–5510.

    Article  CAS  PubMed  Google Scholar 

  52. Groth, A. C., Olivares, E. C., Thyagarajan, B., and Calos, M. P. (2000) A phage integrase directs efficient site-specific integration in human cells. Proc. Natl. Acad. Sci. USA 97, 5995–6000.

    Article  CAS  PubMed  Google Scholar 

  53. Smith, M. C., Till, R., Brady, K., Soultanas, P., and Thorpe, H. (2004) Synapsis and DNA cleavage in phiC31 integrase-mediated site-specific recombination. Nucleic Acids Res. 32, 2607–2617.

    Article  CAS  PubMed  Google Scholar 

  54. Belteki, G., Gertsenstein, M., Ow, D. W., and Nagy, A. (2003) Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase. Nat. Biotechnol. 21, 321–324.

    Article  CAS  PubMed  Google Scholar 

  55. Allen, B. G. and Weeks, D. L. (2005) Transgenic Xenopus laevis embryos can be generated using phiC31 integrase. Nat. Methods 2, 975–979.

    Article  CAS  PubMed  Google Scholar 

  56. Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S., and Calos, M. P. (2001) Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol. Cell Biol. 21, 3926–3934.

    Article  CAS  PubMed  Google Scholar 

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© 2008 Humana Press Inc., Totowa, NJ

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Bischof, J., Basler, K. (2008). Recombinases and Their Use in Gene Activation, Gene Inactivation, and Transgenesis. In: Dahmann, C. (eds) Drosophila. Methods in Molecular Biology, vol 420. Humana Press. https://doi.org/10.1007/978-1-59745-583-1_10

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  • DOI: https://doi.org/10.1007/978-1-59745-583-1_10

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-817-1

  • Online ISBN: 978-1-59745-583-1

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