Gene targeting can result from homologous recombination between an introduced DNA molecule and the homologous genomic locus. In this case, a reciprocal exchange of genetic sequences occurs between the two DNA molecules. Alternatively, it may result from a gene conversion event, which leads to the adaptation of the sequence of one strand to the sequence of another strand. Gene conversion involves local copying of genetic information from one strand to another and is not necessarily associated with cross-overs. A targeted and specificgene inactivation system will facilitate stable and heritable gene silencing with more reliability than conventional systems such as anti-sense RNA and cosuppression.
The ability to inactivate target genes in an efficient manner will also obviate the need for generating large populations of insertionally inactivated plants for each crop to evaluate gene function. Secondly,targeted modification of gene sequences will make in vivo protein engineering a reality, in essence enabling engineering of new genetic variation in a directed and predictable fashion. Thirdly, gene targeting will enable facile replacement and exchange of genes and promoters in the genome. A novel gene could be placed adjacent to a promoter driving a desirable expression pattern in its normal chromosomal context resulting in predictable levels and patterns of gene expression.
This will make conventional transgenic modifications obsolete because the current techniques result in random integration into the genome and wide variation in transgene expression levels due to position effects. The tremendous potential for biological information and biotechnological applications from gene targeting has resulted in concerted efforts to develop the technology for various species. Effective gene targeting methods were first developed in yeast (reviewed in ROTHSTEIN, 1991) and led to an explosion in biological information by facilitating gene knockouts and subtle gene modifications. The value of this technique and resulting information is evident from large-scale application of gene targeting to create a population of yeast mutants with each open reading frame inactivated (WINZELERet al., 1999). Effective gene targeting has also been achieved in mammals enabling functional genomics though gene knockouts
Use of Agrobacterium and T-DNA for gene targeting is an obvious approach with great promise given the availability of Agrobacterium strains capable of infecting all major crop species, and the technical ease of engineering T-DNA molecules. However, successful application of this system to gene targeting is very limited. The general system involves engineering a T-DNA cassette with fragments of DNA homologous to the target genomic locus flanking a disruption cassette, typically a selectable marker. The T-DNA cassette is transferred to plant cells using Agrobacterium and the flanking homology fragments target the T-DNA to the correct genomic locus. A subsequent recombination event transfers the disruption cassette into the plant genome thereby insertionally inactivating the target gene. Using a combination of positive and negative selectable markers true gene targeting events are enriched from the background of random integration of the T-DNA throughout the genome.
However, results to date have been disappointing. Using a strategy of reactivating a defective selectable marker placed in the tobacco genome, OFFRINGA et al. (1990,1993) demonstrated the tenability of T-DNA-mediated gene targeting. But even with a strong selectable phenotype to identify gene targeting events, the frequency of actual gene targeting was very low (-10-7. Targeted inactivation of native genomic loci has been demonstrated in Arabidopsis with frequencies of 11750transgenic lines or 212,580 transgenic calli being obtained (MIAOand LAM, 1995). Gene targeting frequency with T-DNA cassettes can be increased through artificially generating DNA lesions at target loci (PUCHTAet al., 1996),and some increase in gene targeting frequency has been achieved by increasing the recombination potential of plant cells (REISSet al., 2000). However, the low frequency of gene targeting currently achieveable using T-DNA limits application of this technology to genes with an easily selectable or screenable phenotype.
The second successful method of gene targeting in plants utilizes hybrid RNA/DNA molecules to catalyze specific gene conversion events. These molecules consist of two 20-30 bp complementary oligonucleotides, one RNA and the other DNA, which are homologous to the genomic locus of interest except for a single base mismatch (YOONet al., 1996). The gene targeting substrate is transferred to plant cells by particle bombardment. Some of the substrate enters the nucleus, and the homology of the RNA/DNA molecule enables it to pair with the target genomic locus. By an as yet undefined mechanism, the base change encoded by the RNAIDNA hybrid molecule is transferred to the genomic sequence thereby creating a specifically altered gene in vivo.
The genomic base change may be engineered to create a non-sense mutation thereby shutting down functional expression of the gene of interest, or alter a specific amino acid and create a protein with altered biochemical properties. Both of these possibilities have recently been conducted in maize where activity of a selectable marker was altered and a native gene was changed to confer heritable herbicide resistance in regenerated maize plants (ZHUet al., 1999, 2000). However, only a very low frequency was obtained (lop5).Reliance on biolistics to deliver the gene targeting substrate greatly limits exploitation of this technology.The low frequency of gene targeting raises the problem of screening large numbers of calli,before regenerating plants, for the successful targeting event and, therefore, severely limits general application of the technology.
Developing a system that could generate hybrid molecules of large size and effectively deliver them to the nucleus with limited tissue culture steps would greatly improve the application of this technology. At present, applications appear to be limited to modifying genes with easily selectable or screenable phenotypes thereby preventing general application in functional genomics and crop improvement programs. It is to be hoped that further research in design and delivery of gene targeting substrates and increased understanding of mechanisms of DNA recombination and repair in plants will eventually lead to a realistically useful system for targeted gene modifications in plants. Gene targeting will then be a powerful tool for applying information garnered from crop genomics programs to crop improvement.
