In order to understand the role of a given gene product in a given cell type at a given developmental stage, genetic techniques are being developed that allow for the introduction of defined mutations into the mouse genome at will, in a specific cell type and at a chosen time. Most current conditional gene targeting systems are based on the use of the site-specific recombinase Cre which catalyzes recombination between two 34 bp loxP recognition sites.
The basic strategy for Cre/loxP-directed gene knockout experiments is to flank (‘flox’) an essential exon of the gene of interest with two loxP sites (by homologous recombination in ES cells), and then to ‘deliver’ Cre to excise the intervening DNA including the exon from the chromosome, thus generating a null allele in all cells where Cre is active. ‘Delivery’ of Cre can be achieved by crossing mice carrying the ‘floxed’ target gene with transgenic Cre-expressing mice. Clearly, key to successful conditional gene targeting is the availability of Cre transgenic mouse strains in which Cre activity is tightly controlled in space and time.
To this end, we have developed ligand-dependent chimeric Cre recombinases, so-called CreER recombinases. They consist of Cre fused to mutated hormone binding domains of the estrogen receptor. The CreER recombinases are inactive, but can be activated by the synthetic estrogen receptor ligand tamoxifen, therefore allowing for external temporal control of Cre activity. Indeed, by combining tissue-specific expression of a CreER recombinase with its tamoxifen-dependent activity, the excision of ‘floxed’ chromosomal DNA can be controlled both spatially and temporally by administration of tamoxifen to the mouse. Our current efforts are primarily directed towards further refining ligand-activated site-specific recombination in mice. Specifically, we try...
... to increase the efficiency of the system in mice by using novel ‘high-sensitivity’ CreER-like recombinases,
... to develop CreER-based methods to genetically label wild-type and knockout cells and to follow their fate during embryonic development or during disease progression in the adult animal (Cre/lox-assisted cell fate mapping)
... to use spatio-temporally controlled somatic mutagenesis to study the role of selected genes in the cardiovascular and nervous system.