Gene Targeting in Plants

"Gene targeting" is the targeted replacement of an endogenous gene by a precisely manipulated transgene. Because the transgene is directed to a specific sequence in the genome, and integrated via homologous recombination, very precise and sophisticated genetic engineering is possible. This technique is widely used to create novel alleles of genes in yeast, and is also routinely used to generate mutant mice by regeneration of gene-targeted embryonic stem cells. Such is the precision of gene targeting that precise alteration of single bases is possible.

In plants, high-efficiency gene targeting is possible only in the model moss, Physcomitrella patens: an exciting emerging model system for the study of plant cell biology and development. As a bryophyte, it occupies a basal position in the land plant phylogeny, and it is therefore the system of choice for comparative genomic studies of the evolution of gene function. The complete sequence of Physcomitrella is known and a range of genetic and genomic tools are available for functional genomic studies.

Understanding the mechanism of gene targeting in a plant competent to undertake this process is a prerequisite for the development of rational strategies for developing gene targeting in crop species. This project will use a combination of complementary experimental approaches to analyse the mechanism of gene targeting in Physcomitrella.

The principal approach will be to identify components of the homologous recombination-mediated DNA repair pathway, and to construct targeted mutant strains in which the efficiency of gene targeting can be determined. Candidate genes include both evolutionarily conserved components of DNA repair pathways, and novel genes identified by transcriptomic analysis of the DNA damage response. Targeted mutagenesis also allows "knock-in" of epitope- and affinity-tags to specific genes, and these will be used to characterise interactions between members of recombination complexes, in vivo and in vitro.