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PVX-Cre-mediated marker gene elimination from transgenic plants

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Abstract

Cre recombinase gene from bacteriophage P1 was transiently expressed by a Potato Virus X (PVX)-based vector in transgenic lox-target Nicotiana benthamiana plants to remove the selectable marker gene. The target construct consisted of two directly oriented lox sites flanking a bar gene located between a gfpcoding region and an upstream CaMV 35S promoter. The Cre-mediated excision of intervening sequence placed the gfp coding region under the transcriptional control of the CaMV 35S promoter. GFP activity was observed in PVX-Cre systemically infected leaves, regenerants from PVX-Cre infected explants and T 1 progeny of these regenerants. PVX-Cre was removed efficiently from the regenerants by adding the nucleoside analogue ribavirin to the culture medium. Molecular data proved a correlation between gfp expression and precise site-specific excision of the bargene in all examined transgenic lines. The frequency of recombination expressed as a percentage of regenerated plants exhibiting marker gene excision varied from 48% to 82%. These results demonstrate that a plant virus vector can be used efficiently to express cre recombinase in vivo providing an alternative method for the production of transgenic plants without marker genes.

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References

  • Anton, M. and Graham, F. L. 1995. Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression. J. Virol. 69: 4600–4606.

    PubMed  Google Scholar 

  • Austin, S., Ziese, M. and Sternberg, N. 1981. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 25: 729–736.

    PubMed  Google Scholar 

  • Bergemann, J., Kühlcke, K., Fehse, B., Ratz, I., Ostertag, W. and Lother, H. 1995. Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination. Nucl. Acids Res. 23: 4451–4456.

    PubMed  Google Scholar 

  • Baubonis, W. and Sauer, B. 1993. Genomic targeting with purified Cre recombinase. Nucl. Acids Res. 21: 2025–2029.

    PubMed  Google Scholar 

  • Brooks, A. I., Cory-Slechta, D. A. and Federoff, H. J. 2000. Gene-experience interaction alters the cholinergic septohippocampal pathway of mice. Proc. Natl. Acad. Sci. USA 97: 13378–13383.

    PubMed  Google Scholar 

  • Coppoolse, E. R., de Vroomen, M. J., Roelofs, D., Smit, J., van Gennip, F., Hersmus, B. J. M., Nijkamp, H. J. and van Haaren, M. J. J. 2003. Cre recombinase expression can result in phenotypic aberrations in plants. Plant Mol. Biol. 51: 263–279.

    PubMed  Google Scholar 

  • Corneille, S., Lutz, K., Svab, Z. and Maliga, P. 2001. Efficient elimination of selectable marker genes from the plastid genome by the Cre-lox site-specific recombination system. Plant J. 27: 171–178.

    Google Scholar 

  • Dale, E. C. and Ow, D. W. 1990. Intra and intermolecular site specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene 91: 79–85.

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • Dawson, W. O. and Lozoya-Saldana, H. 1984. Examination of the mode of action of ribavirin against tobacco mosaic virus. Intervirology 22: 77–84.

    PubMed  Google Scholar 

  • Esco bar, C., Hernandez, L. E., Jimenez, A., Greissen, G., Ruiz, M. T. and Mullineaux, P. M. 2003. Transient expression of Arabidopsis thaliana ascorbate peroxidase: Nicotiana benthamiana plants infected with recombinant potato virus X. Plant Cell Rep. 21: 699–704.

    PubMed  Google Scholar 

  • De Fazio, G., Caner, J. and Vicente, M. 1978. Inhibitory effect of virazole (ribavirin)on the replication of tomato white necrosis virus (VNBT). Arch. Virol. 58: 153–156.

    PubMed  Google Scholar 

  • De Fazio, G., Caner, J. and Vicente, M. 1980. E. ect of virazole (ribavirin)on tomato spotted wilt virus in two systemic hosts, tomato and tobacco. Arch. Virol. 63: 305–309.

    PubMed  Google Scholar 

  • Gleave, A. P., Mitra, D. S., Mudge, S. R. and Morris, B. A. M. 1999. 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.

    PubMed  Google Scholar 

  • Goldsbrough, A. P., Lastrella, C. N. and Yoder, J. I. 1993. Transposition-mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Biotechnology 11: 1286–1292.

    Google Scholar 

  • Hare, P. D. and Chua, N.-H. 2002. Excision of selectable marker genes from transgenic plants. Nature Biotechnol. 20: 575–580.

    Google Scholar 

  • Hausmann, L. and Töpfer, R. 1999. Entwicklung von Plasmid Vektoren. Vort. Pflanzenzüchtg. 45: 155–172.

    Google Scholar 

  • 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.

    PubMed  Google Scholar 

  • Heim, R., Cubitt, A. B. and Tsien, R. Y. 1995. Improved green fluorescence. Nature 373: 663–664.

    Google Scholar 

  • Hoess, R. H. and Abremski, K. 1985. Mechanism of strand cleavage and exchange in the Crelox site-specific recombination system. J. Mol. Biol. 181: 351–362.

    PubMed  Google Scholar 

  • Hoff, T., Schnorr, K. M. and Mundy, J. 2001. A recombinase-mediated transcriptional induction system in transgenic plants. Plant Mol. Biol. 45: 41–49.

    PubMed  Google Scholar 

  • Hohn, B., Levy, A. A. and Puchta, H. 2001. Elimination of selection markers from transgenic plants. Curr. Opin. Biotechnol. 12: 139–143.

    PubMed  Google Scholar 

  • Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eicholtz, D., Rogers, S. G. and Fraley, R. T. 1985. A simple and general method of transferring genes into plants. Science 277: 1229–1231.

    Google Scholar 

  • Kopertekh, L., Jüttner, G. and Schiemann, J. 2004. Site-specific recombination induced in transgenic plants by PVX virus vector expressing bacteriophage P1 recombinase. Plant Sci. 166: 485–492.

    Google Scholar 

  • Lerch, B. 1987. On the inhibition of plant virus multiplication by ribavirin. Antiviral Res. 7: 257–270.

    PubMed  Google Scholar 

  • Loonstra, A., Vooijs, M., Beverloo, H. B., Allak, B. A., van Drunen, E., Kanaar, R., Berns, A. and Jonkers, J. 2001. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc. Natl. Acad. Sci. USA 98: 9209–9214.

    PubMed  Google Scholar 

  • Lunel, F., Veillon, P., Fouchard-Hubert, I., Loustaud-Ratti, V., Abergel, A., Silvain, C., Rifflet H., Blanchi, A., Causse, X., Bacq, Y. and Payan, C. 2003. Antiviral effect of ribavirin in early non-responders to interferon monotherapy assessed by kinetics of hepatitis C virus RNA and hepatitis C virus core antigen. J. Hepatol. 39: 826–833.

    PubMed  Google Scholar 

  • McCormick, A. A., Reinl, S. J., Cameron, T. I., Vojdani, F., Fronefield, M., Levy, R. and Tuse, D. 2003. Individualized human scFv vaccines produced in plants: humoral antiidiotype responses in vaccinated mice confirm relevance to the tumor Ig. J. Immunol. Meth. 278: 95–104.

    Google Scholar 

  • McKnight, T. D., Lillis, M. T. and Simpson, R. B. 1987. Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant. Mol. Biol. 8: 439–445.

    Google Scholar 

  • Mlynarova, L. and Nap, J.-P. 2003. A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco. Trans Res. 12: 45–57.

    Google Scholar 

  • Mor, T. S., Moon, Y. S., Palmer, K. E. and Mason, H. S. 2003. Geminivirus vectors for high-level expression of foreign proteins in plant cells. Biotechnol. Bioeng. 81: 430–437.

    PubMed  Google Scholar 

  • Pogue, G. P., Lindbo, J. A., Garger, S. J. and Fitzmaurice, W. P. 2002. Making an ally from an enemy: plant virology and the new agriculture. Annu. Rev. Phytopathol. 40: 45–74.

    PubMed  Google Scholar 

  • Puchta, H. 2003. Marker-free transgenic plants. Plant Cell Tissue Organ Cult. 74: 123–134.

    Google Scholar 

  • Raj, S. K., Aminuddin, A. M. and Singh, B. P. 1991. Elimination of eggplant mottled crinkle virus using virazole in explant cultures of Solanum melongena L. Indian J. Exp. Biol. 29: 594–595.

    PubMed  Google Scholar 

  • Saitoh, H., Kiba, A., Nishihara, M., Yamamura, S., Suzuki, K. and Terauchi, R. 2001. Production of antimicrobial defensin in Nicotiana benthamiana with a Potato Virus X vector. MPMI 14: 111–115.

    PubMed  Google Scholar 

  • Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Press, Cold Spring Harbor, NY.

    Google Scholar 

  • Schmidt, E. E., Taylor, D. S., Prigge, J. R., Barnett, S. and Capecchi, M. R. 2000. Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc. Natl. Acad. Sci. USA 97: 13702–13707.

    PubMed  Google Scholar 

  • Shepard, J. F. 1975. Regeneration of plants from protoplasts of Potato Virus X-infected tobacco leaves. Virology 78: 261–266.

    Google Scholar 

  • Silver, D. P. and Livingston, D. M. 2001. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol. Cell. 8: 233–243.

    PubMed  Google Scholar 

  • Timmermans, M. C. P., Maliga, P., Vieira, J. and Messing, J. 1990. The pFF plasmids: cassettes utilising CaMV sequences for expression of foreign genes in plants. J. Biotechnol. 14: 333–344.

    PubMed  Google Scholar 

  • Töpfer, R., Maas, C., Horicke-Grandpierre, C., Schell, J. and Steinbiss, H. H. 1993. Expression vectors for high-level gene expression in dicotyledonous and monocotyledonous plants. Meth. Enzymol. 217: 67–87.

    PubMed  Google Scholar 

  • Vergunst, A. C., Schrammeijer, B., den Dulk-Ras, A., de Vlaam, C. M. T., Regensburg-Tuink, T. J. G. and Hooykaas, P. J. J. 2000. Vir B/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290: 979–982.

    PubMed  Google Scholar 

  • Vieira, J. and Messing, J. 1982. The pUC plasmids, an M13mp7derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259–268.

    PubMed  Google Scholar 

  • Vo, N. V., Young, K. C. and Lai, M. M. 2003. Mutagenic and inhibitory effects of ribavirin on hepatitis C virus R polymerase. Biochemistry 42: 10462–10471.

    PubMed  Google Scholar 

  • Wang, Y., Krushel, L. A. and Edelman, G. M. 1996. Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene. Proc. Natl. Acad. Sci. USA 93: 3932–3936.

    PubMed  Google Scholar 

  • Westerman, K. A. and Leboulch, P. 1996. Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc. Natl. Acad. Sci. USA 93: 8971–8976.

    PubMed  Google Scholar 

  • Wigdorovitz, A., Perez Filgueria, D. M., Robertson, N., Carrilo, C., Sadir, A. M., Morris, T. J. and Borca, M. V. 1999. Protection of mice against challenge with foot and mouth disease virus (FMDV)by immunisation with folia extracts from plants infected with recombinant Tobacco Mosaic Virus expressing the FMDV structural protein VP1. Virology 264: 85–91.

    PubMed  Google Scholar 

  • Will, E., Klump, H., Heffner, N., Schwieger, M. and Schiedlmeier, B. 2002. Unmodified Cre recombinase crosses the membrane. Nucl. Acids Res. 30: e59.

    PubMed  Google Scholar 

  • Yoder, J. I. and Goldsbrough, A. P. 1994. Transformation systems for generating marker-free transgenic plants. Bio/ Technology 12: 263–267.

    Google Scholar 

  • Zuo, J., Niu, Q.-W., Moller, S. G. and Chua, N.-H. 2001. Chemical-regulated, site-specific DNA excision in transgenic plants. Nat. Biotechnol. 19: 157–161.

    PubMed  Google Scholar 

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  1. Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biosafety, Messeweg, 11-12, Germany

    L. Kopertekh, G. Jüttner & J. Schiemann

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  1. L. Kopertekh
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  2. G. Jüttner
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  3. J. Schiemann
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Kopertekh, L., Jüttner, G. & Schiemann, J. PVX-Cre-mediated marker gene elimination from transgenic plants. Plant Mol Biol 55, 491–500 (2004). https://doi.org/10.1007/s11103-004-0237-8

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