title: 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 beta (GSK-3beta) gene are fused to an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3beta, producing a severe loss-of-function allele. In the presence of C20-MaRap, a highly specific, nontoxic, cell-permeable small molecule, GSK-3betaFRB* binds to the ubiquitously expressed FKBP12 protein. This interaction stabilizes GSK-3betaFRB* 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.
authors.affiliation: Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Kryn
authors.initials: K
authors.lastname: Stankunas
authors.forename: J Henri
authors.initials: JH
authors.lastname: Bayle
authors.forename: Jason E
authors.initials: JE
authors.lastname: Gestwicki
authors.forename: Yun-Ming
authors.initials: YM
authors.lastname: Lin
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wandless
authors.forename: Gerald R
authors.initials: GR
authors.lastname: Crabtree
mesh_terms: D000483:Alleles; D000818:Animals; D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism; D019281:Dimerization; D004622:Embryo, Mammalian / Q000502:physiology; D004795:Enzyme Stability; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D051379:Mice; D008822:Mice, Transgenic; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000378:metabolism; D011247:Pregnancy; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020123:Sirolimus / Q000737:chemistry / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D020123:Sirolimus