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Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

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Abstract

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.

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Abbreviations

CaMV 35S:

Cauliflower mosaic virus 35S

cDNA:

Complementary DNA

cox:

Cytochrome oxidase

Ct:

Cycle threshold

CTAB:

Cetyl trimethyl ammonium bromide

EDTA:

Ethylenediaminetetraacetic acid

hsp70:

Heat-shock protein 70

LB:

Luria-Bertani

loxP:

Locus of X-ing over

MCS:

Multiple cloning site

MS:

Murashige and Skoog

M-MuLV:

Moloney murine leukemia virus

NaCl:

Sodium clhoride

nptII:

Neomycin phosphotransferase II

NOS:

Nopaline syntase

T-DNA:

Transferred DNA

TBE:

Tris-borate-ethylenediaminetetraacetic acid

pCIP:

Plasmid International Potato Center

PCR:

Polymerase chain reaction

RT-qPCR:

Real time-quantitative polymerase chain reaction

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Acknowledgements

We are grateful to Dr. B. Kisters-Woike from Institute of Genetics of the University of Cologne, Germany who provide the plasmid pET11a-cre-wt containing the cre recombinase gene, to Dr. Th. Schmülling from the University of Tuebingen, Germany who provide the plasmid pHSIPT containing the hsp70 promoter of Drosophila melanogaster, and to Dr. P.J.M. van den Elzen at Mogen Intl., Leiden, The Netherlands for providing the plasmid pMOG800. This research was partially supported by a grant from the Rockefeller Foundation, Food security program.

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Author notes

  1. Wilmer Cuellar

    Present address: Department of Applied Biology, University of Helsinki, P.O. Box 27, Latokartanonkaari 7, FIN-00014, Helsinki, Finland

  2. Armando Casas

    Present address: Plant Science Initiative, E209 Beadle Center, University of Nebraska-Lincoln, 1901 Vine Street, Lincoln, NE68588, USA

  3. Luis Ñopo

    Present address: Arkansas Biosciences Institute, Arkansas State University, 504 University Loop East, Jonesboro, AR, 72401, USA

  4. Prakash Chudalayandi

    Present address: Department of Cell Biology and Physiology, Washington University, St Louis, MO, 63110, USA

Authors and Affiliations

  1. Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, Lima, 12, Peru

    Wilmer Cuellar, Amélie Gaudin, Dennis Solórzano, Armando Casas, Luis Ñopo, Prakash Chudalayandi, Giuliana Medrano, Jan Kreuze & Marc Ghislain

  2. Universidad Nacional Frederico Villarreal, Calle San Marcos 351, Pueblo Libre, Lima, Peru

    Wilmer Cuellar & Dennis Solórzano

  3. Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), P.O. Box 7080, SE-750 07, Uppsala, Sweden

    Jan Kreuze

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  1. Wilmer Cuellar
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Correspondence to Marc Ghislain.

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Cuellar, W., Gaudin, A., Solórzano, D. et al. Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato. Plant Mol Biol 62, 71–82 (2006). https://doi.org/10.1007/s11103-006-9004-3

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  • DOI: https://doi.org/10.1007/s11103-006-9004-3

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