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Chemical-regulated, site-specific DNA excision in transgenic plants

Nature Biotechnology volume 19, pages 157–161 (2001)Cite this article

Abstract

We have developed a chemical-inducible, site-specific DNA excision system in transgenic Arabidopsis plants mediated by the Cre/loxP DNA recombination system. Expression of the Cre recombinase was tightly controlled by an estrogen receptor-based fusion transactivator XVE. Upon induction by β-estradiol, sequences encoding the selectable marker, Cre, and XVE sandwiched by two loxP sites were excised from the Arabidopsis genome, leading to activation of the downstream GFP (green fluorescent protein) reporter gene. Genetic and molecular analyses indicated that the system is tightly controlled, showing high-efficiency inducible DNA excision in all 19 transgenic events tested with either single or multiple T-DNA insertions. The system provides a highly reliable method to generate marker-free transgenic plants after transformation through either organogenesis or somatic embryogenesis.

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Figure 1: A schematic diagram of the CLX vector and β-estradiol-induced DNA excision.
Figure 2: Molecular characterization of β-estradiol-induced site-specific DNA excision in transgenic Arabidopsis plants.

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References

  1. Yoder, J.I. & Goldsbrough, A.P. Transformation systems for generating marker free transgenic plants. Bio/Technology 12, 263–267 (1994).

    Article  CAS  Google Scholar 

  2. Ow, D.W. & Medberry, S.L. Genome manipulations through site-specific recombination. Crit. Rev. Plant Sci. 14, 239–261 (1995).

    Article  CAS  Google Scholar 

  3. Dale, E.C. & Ow, D.W. Gene transfer with subsequent removal of the selection gene from the host genome. Proc. Natl. Acad. Sci. USA 88, 10558–10562 (1991).

    Article  CAS  Google Scholar 

  4. Russell, S.H., Hoopes, J.L. & Odell, J.T. Directed excision of a transgene from the plant genome. Mol. Gen. Genet. 234, 49–59 (1992).

    CAS  Google Scholar 

  5. Bayley, C.C., Morgan, M., Dale, E.C. & Ow, D.W. Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system. Plant Mol. Biol. 18, 353–361 (1992).

    Article  CAS  Google Scholar 

  6. Qin, M., Bayley, C., Stockton, T. & Ow, D.W. Cre recombinase-mediated site-specific recombination between plant chromosomes. Proc. Natl. Acad. Sci. USA 91, 1706–1710 (1994).

    Article  CAS  Google Scholar 

  7. Lloyd, A.M. & Davis, R.W. Functional expression of the yeast FLP/FRT site-specific recombination system in Nicotiana tabacum. Mol. Gen. Genet. 242, 653–657 (1994).

    Article  CAS  Google Scholar 

  8. Komari, T., Hiei, Y., Saito, Y., Murai, N. & Kumashiro, T. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J. 10, 165–174 (1996).

    Article  CAS  Google Scholar 

  9. Gleave, A.P., Mitra, D.S., Mudge, S.R. & Morris, B.A. Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol. Biol. 40, 223–235 (1999).

    Article  CAS  Google Scholar 

  10. Barry, G.F., Rogers, S.G., Fraley, R.T. & Brand, L. Identification of a cloned cytokinin biosynthetic gene. Proc. Natl. Acad. Sci. USA 81, 4776–4780 (1984).

    Article  CAS  Google Scholar 

  11. Akiyoshi, D.E., Klee, H., Amasino, R.M., Nester, E.W. & Gordon, M.P. T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc. Natl. Acad. Sci. USA 81, 5994–5998 (1984).

    Article  CAS  Google Scholar 

  12. Ebinuma, H., Sugita, K., Matsunaga, E. & Yamakado, M. Selection of marker-free transgenic plants using the isopentenyl transferase gene as a selectable marker. Proc. Natl. Acad. Sci. USA 94, 2117–2121 (1997).

    Article  CAS  Google Scholar 

  13. Sugita, K., Kasahara, T., Matsunaga, E. & Ebinuma, H. A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency. Plant J. 22, 461–469 (2000).

    Article  CAS  Google Scholar 

  14. Kunkel, T., Niu, Q.-W., Chan, Y.S. & Chua, N.-H. Inducible isopentenyl transferase as a high-efficiency marker for plant transformation. Nat. Biotechnol. 17, 916–919 (1999).

    Article  CAS  Google Scholar 

  15. Aoyama, T. & Chua, N.-H. A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 11, 605–612 (1997).

    Article  CAS  Google Scholar 

  16. Zubko, E., Scutt, C. & Meyer, P. Intrachromosomal recombination between two attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat. Biotechnol. 18, 442–445 (2000).

    Article  CAS  Google Scholar 

  17. Satina, S., Blakeslee, A.F. & Avery, A.G. Demonstration of the three germ layers in the shoot apex of Datura by means of induced polyploidy in periclinal chimeras. Am. J. Bot. 27, 895–905 (1940).

    Article  Google Scholar 

  18. Irish, V.F. & Sussex, I. M. A fate map of the Arabidopsis embryonic shoot apical meristem. Development 115, 745–753 (1992).

    Google Scholar 

  19. Furner, I.J. & Pumfrey, J.E. Cell fate in the shoot apical meristem of Arabidopsis thaliana. Development 115, 755–764 (1992).

    Google Scholar 

  20. Marcotrigiano, M. & Bernatzky, R. Arrangement of cell layers in the shoot apical meristems of periclinal chimeras influences cell fate. Plant J. 7, 193–202 (1995).

    Article  Google Scholar 

  21. Zuo, J., Niu, Q.-W. & Chua, N.-H. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265–273 (2000).

    Article  CAS  Google Scholar 

  22. Austin, S., Ziese, M. & Sternberg, N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 25, 729–736 (1981).

    Article  CAS  Google Scholar 

  23. Odell, J., Caimi, P., Sauer, B. & Russell, S. Site-directed recombination in the genome of transgenic tobacco. Mol. Gen. Genet. 223, 369–378 (1990).

    Article  CAS  Google Scholar 

  24. Zuo, J. et al. KORRIGAN, an Arabidopsis endo-1,4-β-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12, 1137–1152 (2000).

    CAS  Google Scholar 

  25. Ishige, F., Takaichi, M., Foster, R., Chua, N.-H. & Oeda, K. A G-box motif (GCCACGTGCC) tetramer confers high-level constitutive expression in dicot and monocot plants. Plant J. 18, 443–448 (1999).

    Article  CAS  Google Scholar 

  26. Koncz, C. et al. High-frequency T-DNA-mediated gene tagging in plants. Proc. Natl. Acad. Sci. USA 86, 8467–8471 (1989).

    Article  CAS  Google Scholar 

  27. Murashige, T. & Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497 (1962).

    Article  CAS  Google Scholar 

  28. Sambrook, J., Fritsch, E.F. & Manistis, T. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1989).

    Google Scholar 

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Acknowledgements

We thank Dr. D. Ow for providing pMM23. We are grateful to anonymous reviewers for their valuable comments.

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Author notes

  1. Jianru Zuo and Qi-Wen Niu: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, 10021, NY

    Jianru Zuo, Qi-Wen Niu, Simon Geir Møller & Nam-Hai Chua

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  1. Jianru Zuo
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  2. Qi-Wen Niu
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  3. Simon Geir Møller
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  4. Nam-Hai Chua
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Correspondence to Nam-Hai Chua.

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Zuo, J., Niu, QW., Møller, S. et al. Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnol 19, 157–161 (2001). https://doi.org/10.1038/84428

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