Published in Probe Volume 4(3-4): August 1994-January 1995
Jim Wallis and Dan Guerra
Department of Microbiology, Molecular Biology,
and Biochemistry, University of Idaho
Moscow, Idaho 83844
Early advances in the alteration of plant genomes by application of recombinant DNA technology were not directed to commercial crop varieties. However, many crop plants have now been stably transformed with useful genes and their progeny examined for inheritance of the introduced traits.
Truly successful improvement of crop plants via recombinant DNA technology has been limited both by the methodology of introducting new or altered genes into plants, and by the difficulties of regenerating whole plants after the necessary manipulative steps.
There are other problems associated with using this new technology in crop improvement programs. Current methods of gene transfer all introduce the DNA as random insertions. Such random insertions can result in undesirable changes in phenotype or in a loss of control over the expression of the transgene. In addition, current gene transfer techniques depend on the co-introduction of a selectable antibiotic marker along with the transgenes of interest, which are typically non-selectable. Further problems arise because we have not yet perfected the ability to target new genetic material to a specific locus in the plant genome.
One method of addressing these problems is by use of a site-specific DNA recombinase. Progress has been made using both the cre recombinase, a product of lambda phage in Escherichia coli, and the FLP recombinase, an enzyme native to the 2 micron plasmid of Saccharomyces cerevisiae.
These recombinases alter the arrangement of DNA sequences in very specific ways. The FLP recombinase is active at a particular 34 base pair DNA sequence, termed the FRT (FLP recombinase target) sequence. When two of these FRT sites are present, the FLP enzyme creates double-stranded breaks in the DNA strands, exchanges the ends of the first FRT with those of the second target sequence, and then reattaches the exchanged strands. This process leads to inversion or deletion of the DNA which lies between the two sites. Whether there is an inversion or deletion depends on the orientation of the FRT sites: if the sites are in the same direction, the intervening DNA will be deleted, but if the sites are in opposite orientation, the DNA is inverted.
The FLP recombinase is currently being used to genetically engineer crop species. One immediate goal is to use the recombinase to remove antibiotic resistance genes in transgenic plants. Since there is currently public concern over the presence of such antibiotic resistance markers, removal of the resistance gene will improve the marketability of transgenic plant products. Marker removal will also permit use of the original selectable marker to introduce other useful genes into these engineered plants. The FRT site that remains after resistance marker deletion will also provide a means for multiple subsequent insertions of transgenes into a single site in the genome. Introduction of new genes at specific locations may avoid the phenotypic irregularities and problems with expression that result from random integration.
Finally, the FLP recombinase could be used as a negative selectable marker for experiments to replace genes by homologous recombination. An antibiotic resistance marker flanked by FRT sites and regions homologous to the crop plant genome would be deleted if a more distant recombinase gene was inserted in the genome by illegitimate recombination; homologous recombinants would not contain the FLP coding sequence and would retain antibiotic resistance.
Genetic manipulation by means of this eukaryotic recombinase will mark a significant advance in the biotechnological improvement of crop plants.