Site-directed recombination in the genome of transgenic tobacco |
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Authors: | Joan Odell Perry Caimi Brian Sauer and Sandra Russell |
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Institution: | (1) Agricultural Biotechnology, Agricultural Products Department, Experimental Station, E.I. DuPont de Nemours and Co, 19880-0402 Wilmington, Delaware, USA;(2) Molecular Biology, Central Research and Development Department, Experimental Station, E.I. DuPont de Nemours and Co, 19880-0402 Wilmington, Delaware, USA |
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Abstract: | Summary The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase 11 gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%–100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2–3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression. |
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Keywords: | Site-specific recombination loxP-Cre Plant transformation Sulfonylurea resistance marker |
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