"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.
The Leeds Physcomitrella laboratory is a founder member of the International Physcomitrella Genome Consortium, and is supported by BBSRC funding.
This studentship is supported as part of a BBSRC Doctoral Training Grant to the Centre for Plant Sciences.
Kamisugi Y, Cuming AC, Cove DJ (2005) Parameters determining the efficiency of homologous recombination mediated gene targeting in the moss Physcomitrella patens. Nucleic Acids Res. 33: e173 (http://nar.oxfordjournals.org/cgi/content/full/33/19/e173)
Kamisugi Y, Schlink K, Rensing SA, Schween G, von Stackelberg M, Cuming AC, Reski R, Cove DJ (2006) The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration. Nucleic Acids Res. 34: 6205-6214 (http://nar.oxfordjournals.org/cgi/content/full/34/21/6205)
Rensing SA et al (2008) The Physcomitrella genome reveals insights into the conquest of land by plants. Science: 319: 64-69
Kamisugi Y, von Stackelberg M, Lang D, Care M, Reski R, Rensing SA, Cuming AC (2008) A sequence-anchored genetic linkage map for the moss Physcomitrella patens. Plant J: 56: 855-866 (http://www3.interscience.wiley.com/cgi-bin/fulltext/120848084/PDFSTART)
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