Site-directed recombination in the genome of transgenic tobacco

Abstract

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.

Seed-Specific Gene Activation Mediated by the Cre/lox Site-Specific Recombination System

“…A linker containing XhoI and NcoI sitw was added between the Asp718 and XbaI sites located 3' to the Cg/P. The lox-polyA-lox fragment described by Odell et al (1990) was added as a XhoI-Ncol fragment, after an NcoI linker was added at the HindIII site at the 3' end. This fragnient contains the polyadenylation signal sequence region frcm a tobacco Rubisco small subunit gene (Mazur and Chui, 15185) between directly oriented lox sites.…”

“…Integration between target sites on separate DNA molecules can also occur. The site-specific recombination systems expressed in plants thus far include FLP/FRT (recombinase/recognition site) of the Saccharomyces cerevisiae 2p plasmid (Lyznik et al, 1993;Lloyd and Davis, 1994), R/RS of the Zygosaccharomyces rouxii R1 plasmid (Onouchi et al, 1991), Gin/& of bacteriophage Mu (Maeser and Kahmann, 1991), and Crellox of bacteriophage P1 (Dale and Ow, 1990;Odell et al, 1990); all systems are reviewed by Odell and Russell (1994).…”

“…A lox-bounded coding region placed in inverted orientation with respect to the regulating promoter was expressed after inversion (Dale and Ow,19.90). A loxbounded fragment placed between a promoter and coding region, disrupting gene expression, was excised to restore activity (Odell et al, 1990;Bayley et al, 1992). Translocation between lox sites located on separate chromosomes also brought together a promoter and coding region, resulting in gene activation (Qin et al, 1994).…”

A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre—lox

“…This reaction occurs efficiently in planta (Bayley et aL, 1992;Odell et al, 1990;Qin et al, 1994;Russell et al, 1992). Thus, Dslox transposition moves a Iox site about the genome, and we have demonstrated that Cre recombinase will act on these sites to generate chromosomal rearrangements.…”

“…Previous experiments have shown that Cre recombinase expressed from within a T-DNA can catalyze Iox-/ox recombination efficiently when the Iox sites are integrated together at a second T-DNA locus (Bayley eta/., 1992;Odell et al, 1990;Russell et al, 1992). These experiments have also demonstrated that the rearrangements can be germinally transmitted, as expected.…”