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Transgenic Xenopus laevis embryos can be generated using φC31 integrase

Nature Methods volume 2, pages 975–979 (2005)Cite this article

Abstract

Bacteriophage φC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA. Here we show that the coinjection of mRNA encoding φC31 integrase with plasmid DNA encoding the green fluorescent protein (GFP) can be used to generate transgenic X. laevis embryos. Despite integration into the genome, appropriate promoter expression required modification of the reporter plasmid by bracketing the GFP reporter gene with tandem copies of the chicken β-globin 5′ HS4 insulator to relieve silencing owing to chromatin position effects. These experiments demonstrate that the integration of insulated gene sequences using φC31 integrase can be used to efficiently create transgenic embryos in X. laevis and may increase the practical use of φC31 integrase in other systems as well.

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Figure 1: Coinjection of φC31 integrase with CMV-gfp reporter plasmid allows detection of GFP expression in stage 46 Xenopus embryos.
Figure 2: Southern blot analysis of treated embryos indicates insertion of the CMV-gfp reporter plasmid into the embryonic genome.
Figure 3: Coinjection of φC31 integrase mRNA with insulated CMV-gfp or crystallin lens–gfp reporter plasmid generated Xenopus embryos with tissue-appropriate expression.

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References

  1. Kroll, K.L. & Amaya, E. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173–3183 (1996).

    CAS  PubMed  Google Scholar 

  2. Sparrow, D.B., Latinkic, B. & Mohun, T.J. A simplified method of generating transgenic Xenopus. Nucleic Acids Res. 28, E12 (2000).

    Article  CAS  Google Scholar 

  3. Thorpe, H.M., Wilson, S.E. & Smith, M.C. Control of directionality in the site-specific recombination system of the Streptomyces phage φC31. Mol. Microbiol. 38, 232–241 (2000).

    Article  CAS  Google Scholar 

  4. Thorpe, H.M. & Smith, M.C. 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 (1998).

    Article  CAS  Google Scholar 

  5. Lutz, K.A., Corneille, S., Azhagiri, A.K., Svab, Z. & Maliga, P. A novel approach to plastid transformation utilizes the φC31 phage integrase. Plant J. 37, 906–913 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Chalberg, T.W., Genise, H.L., Vollrath, D. & Calos, M.P. φC31 integrase confers genomic integration and long-term transgene expression in rat retina. Invest. Ophthalmol. Vis. Sci. 46, 2140–2146 (2005).

    Article  Google Scholar 

  8. Thyagarajan, B., Olivares, E.C., Hollis, R.P., Ginsburg, D.S. & Calos, M.P. Site-specific genomic integration in mammalian cells mediated by phage φC31 integrase. Mol. Cell. Biol. 21, 3926–3934 (2001).

    Article  CAS  Google Scholar 

  9. Thomason, L.C., Calendar, R. & Ow, D.W. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage φC31 site-specific recombination system. Mol. Genet. Genomics 265, 1031–1038 (2001).

    Article  CAS  Google Scholar 

  10. Olivares, E.C. et al. Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat. Biotechnol. 20, 1124–1128 (2002).

    Article  CAS  Google Scholar 

  11. Held, P.K. et al. In vivo correction of murine hereditary tyrosinemia type I by φC31 integrase–mediated gene delivery. Mol. Ther. 11, 399–408 (2005).

    Article  CAS  Google Scholar 

  12. Belteki, G., Gertsenstein, M., Ow, D.W. & Nagy, A. Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase. Nat. Biotechnol. 21, 321–324 (2003).

    Article  CAS  Google Scholar 

  13. Groth, A.C., Fish, M., Nusse, R. & Calos, M.P. Construction of transgenic Drosophila by using the site-specific integrase from phage φC31. Genetics 166, 1775–1782 (2004).

    Article  CAS  Google Scholar 

  14. Vize, P.D., Melton, D.A., Hemmati-Brivanlou, A. & Harland, R.M. Assays for gene function in developing Xenopus embryos. Methods Cell Biol. 36, 367–387 (1991).

    Article  CAS  Google Scholar 

  15. Newport, J. & Kirschner, M. A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell 30, 687–696 (1982).

    Article  CAS  Google Scholar 

  16. Kuhn, E.J. & Geyer, P.K. Genomic insulators: connecting properties to mechanism. Curr. Opin. Cell Biol. 15, 259–265 (2003).

    Article  CAS  Google Scholar 

  17. West, A.G., Gaszner, M. & Felsenfeld, G. Insulators: many functions, many mechanisms. Genes Dev. 16, 271–288 (2002).

    Article  Google Scholar 

  18. Bell, A.C. & Felsenfeld, G. Stopped at the border: boundaries and insulators. Curr. Opin. Genet. Dev. 9, 191–198 (1999).

    Article  CAS  Google Scholar 

  19. Chung, J.H., Whiteley, M. & Felsenfeld, G. A 5′ element of the chicken β-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74, 505–514 (1993).

    Article  CAS  Google Scholar 

  20. Pikaart, M.J., Recillas-Targa, F. & Felsenfeld, G. Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev. 12, 2852–2862 (1998).

    Article  CAS  Google Scholar 

  21. Offield, M.F., Hirsch, N. & Grainger, R.M. The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos. Development 127, 1789–1797 (2000).

    CAS  PubMed  Google Scholar 

  22. Smith, M.C., Burns, R.N., Wilson, S.E. & Gregory, M.A. The complete genome sequence of the Streptomyces temperate phage straight φC31: evolutionary relationships to other viruses. Nucleic Acids Res. 27, 2145–2155 (1999).

    Article  CAS  Google Scholar 

  23. Hollis, R.P. et al. Phage integrases for the construction and manipulation of transgenic mammals. Reprod. Biol. Endocrinol. 1, 79–89 (2003).

    Article  Google Scholar 

  24. Rebagliati, M.R., Weeks, D.L., Harvey, R.P. & Melton, D.A. Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell 42, 769–777 (1985).

    Article  CAS  Google Scholar 

  25. Dagle, J.M., Walder, J.A. & Weeks, D.L. Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis. Nucleic Acids Res. 18, 4751–4757 (1990).

    Article  CAS  Google Scholar 

  26. Nieuwkoop, P.D. & Faber, J. Normal table of Xenopus laevis (Daudin): a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. (Garland Pub., New York, 1994).

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Acknowledgements

We thank M. Calos, G. Felsenfeld and P. Krieg for their generosity in providing plasmids used in this study. We thank H. Bartlett, J. Dagle, S. Kolker, C. Fett, E. Hornick and E. Stokasimov for helpful discussions. Funding to D.L.W. from the National Institutes of Health (GM069944 and DC007481) supported this work. B.G.A. is a student in the Medical Scientist Training Program at the Roy J. and Lucille A. Carver College of Medicine, University of Iowa.

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  1. Department of Biochemistry, Bowen Science Building, University of Iowa, Iowa City, 52242, Iowa, USA

    Bryan G Allen & Daniel L Weeks

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  1. Bryan G Allen
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Correspondence to Daniel L Weeks.

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B.G.A. and D.L.W. are authors of a provisional patent application that has been filed related to the use of insulated genes and attB contained in plasmids.

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Allen, B., Weeks, D. Transgenic Xenopus laevis embryos can be generated using φC31 integrase. Nat Methods 2, 975–979 (2005). https://doi.org/10.1038/nmeth814

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