Site-directed recombination in the genome of transgenic tobacco.
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 II 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.
Odell
J
J
Agricultural Products Department, E.I. DuPont de Nemours and Co., Wilmington, Delaware 19880-0402.
Caimi
P
P
Sauer
B
B
Russell
S
S
D015152:Blotting, Northern; D015139:Blotting, Southern; D002678:Chimera / Q000235:genetics; D003433:Crosses, Genetic; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D017343:Genes, Plant; D006579:Heterozygote; D006720:Homozygote; D019426:Integrases; D019868:Kanamycin Kinase; D007613:Kanamycin Resistance / Q000235:genetics; D010641:Phenotype; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D010947:Plants, Toxic; D011995:Recombination, Genetic; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic; D014764:Viral Proteins
D014764:Viral Proteins; D010770:Phosphotransferases; D019868:Kanamycin Kinase; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases