Conditional Protein Alleles Using Knockin Mice and a Chemical Inducer of Dimerization

Abstract

We have developed a general method of making conditional alleles that allows the rapid and reversible regulation of specific proteins. A mouse line was produced in which proteins encoded by the endogenous glycogen synthase kinase-3 β (GSK-3β) gene are fused to an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3β, producing a severe loss-of-function allele. In the presence of C20-MaRap, a highly specific, nontoxic, cell-permeable small molecule, GSK-3βFRB* binds to the ubiquitously expressed FKBP12 protein. This interaction stabilizes GSK-3βFRB* and restores both protein levels and activity. C20-MaRap-mediated stabilization is rapidly reversed by the addition of an FKBP12 binding competitor molecule. This technology may be applied to a wide range of FRB*-tagged mouse genes while retaining their native transcriptional control. Inducible stabilization could be valuable for many developmental and physiological studies and for drug target validation.