LOFT should be generally applicable to mouse genes, including the growing numbers of genes already floxed in the latter case, only the trapped alleles need to be generated to confer reversibility to the pre-existing cKO models. More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO. The floxed and trapped alleles can typically be generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT sites, and can be used independently to achieve conditional and constitutive gene KO, respectively.
This method involves two alleles of a target gene: a standard floxed allele, and a multi-functional allele bearing an FRT-flanked gene-trap cassette, which inactivates the target gene while reporting its expression with green fluorescent protein (GFP) the trapped allele is thus a null and GFP reporter by default, but is convertible into a wild-type allele. We describe a simple method we have named LOFT, which is capable of reversible cKO and free of the limitations associated with existing techniques. A number of methods have been developed to overcome this, but each method has its own limitations. However, a major limitation of this method is that gene KO is not reversible. Conditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice.
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