caa a22 4500 467895368 CHVBK 20180406152802.0 cr unu---uuuuu 170328e20060901xx s 000 0 eng 10.1007/s11103-006-9004-3 doi (NATIONALLICENCE)springer-10.1007/s11103-006-9004-3 Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato [Elektronische Daten] [Wilmer Cuellar, Amélie Gaudin, Dennis Solórzano, Armando Casas, Luis Ñopo, Prakash Chudalayandi, Giuliana Medrano, Jan Kreuze, Marc Ghislain] 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. Springer Science+Business Media B.V., 2006 Cre- loxP recombination system nationallicence Gene excision nationallicence Selectable marker nationallicence Solanum tuberosum nationallicence Transgenic potato nationallicence CaMV 35S : Cauliflower mosaic virus 35S nationallicence cDNA : Complementary DNA nationallicence cox : Cytochrome oxidase nationallicence Ct : Cycle threshold nationallicence CTAB : Cetyl trimethyl ammonium bromide nationallicence EDTA : Ethylenediaminetetraacetic acid nationallicence hsp70 : Heat-shock protein 70 nationallicence LB : Luria-Bertani nationallicence loxP : Locus of X-ing over nationallicence MCS : Multiple cloning site nationallicence MS : Murashige and Skoog nationallicence M-MuLV : Moloney murine leukemia virus nationallicence NaCl : Sodium clhoride nationallicence npt II : Neomycin phosphotransferase II nationallicence NOS : Nopaline syntase nationallicence T-DNA : Transferred DNA nationallicence TBE : Tris-borate-ethylenediaminetetraacetic acid nationallicence pCIP : Plasmid International Potato Center nationallicence PCR : Polymerase chain reaction nationallicence RT-qPCR : Real time-quantitative polymerase chain reaction nationallicence Cuellar Wilmer Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Gaudin Amélie Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Solórzano Dennis Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Casas Armando Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Ñopo Luis Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Chudalayandi Prakash Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Medrano Giuliana Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Kreuze Jan Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Ghislain Marc Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut Plant Molecular Biology Kluwer Academic Publishers 62/1-2(2006-09-01), 71-82 0167-4412 62:1-2<71 2006 62 11103 https://doi.org/10.1007/s11103-006-9004-3 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11103-006-9004-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Cuellar Wilmer Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 700 1- Gaudin Amélie Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 700 1- Solórzano Dennis Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 700 1- Casas Armando Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 700 1- Ñopo Luis Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 700 1- Chudalayandi Prakash Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 700 1- Medrano Giuliana Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 700 1- Kreuze Jan Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 700 1- Ghislain Marc Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement Division, International Potato Center CIP, P.O. Box 1558, 12, Lima, Peru aut NATIONALLICENCE 773 0- Plant Molecular Biology Kluwer Academic Publishers 62/1-2(2006-09-01), 71-82 0167-4412 62:1-2<71 2006 62 11103 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer