Site-Specific DNA Recombinases

Recombinase systems are used to remove no longer needed selection markers when creating knock-in or allelic series models and/or to create conditional tissue- or cell-specific mutations. The two most common systems are Cre recombinase from bacteriophage P1 and FLP recombinase from yeast (Saccharomyces cerevisiae). Both Cre and FLP recombinase catalyze an irreversible, site-specific DNA recombination event between two 34-bp recognition sites, loxP and FRT, respectively. The 34-bp loxP and FRT recognition sites consist of two 13-bp inverse repeats flanking an 8-bp core sequence; the core sequences confer directionality.

Mutations are then created conditionally by expressing the appropriate recombinase in a particular tissue or during a specific time period (i.e., during embryonic development). Recombinase systems can also be used to mis-express genes spatially or temporally, for example during embryogenesis or ontogeny, when expression of the recombinase deletes stop codon sequences thus allowing transcription of a particular gene of interest. Recombinase-mediated recombination is most commonly accomplished using transgenic deletor mouse strains that express Cre/FLP in a tissue specific manner. Recombinases can also be delivered locally, i.e. blown into the lung or injected into a muscle, to modulate gene expression.

Recombinase systems are also used to delete intron-placed marker genes. Removal of the selection marker allows the targeting of yet another gene in a previously targeted ES cell clone. It has also been reported that the intronic PGK-neo marker gene, if left intact, can act as a hypomorphic allele (ref). Reduced gene expression resulted from improper RNA slicing into a cryptic acceptor site within the PGK-neo gene.

Use of a combination of both the Cre/loxP and FLPe/FRT systems in targeting vector design is highly recommended if considering a 3-loxP design. Inclusion of a combination of these elements in the targeting vector results in more straightforward and efficient deletion when creating conditional mutations and allows greater versatility in the use of the mice.