STOCK Gsk3b^tm1Grc/J

Stock number: 017693
Research areas: Developmental Biology Research, Internal/Organ Research, Research Tools

Description

Mice homozygous for this Gsk3b conditional mutation express greatly reduced levels of a destabilized protein. When treated with rapamycin, functional protein expression is restored. This strain may be useful in studies of diabetes, inflammation, cardiovascular disease, cancer, neurodegenerative disease, and bipolar disorder.

Detailed description

In this Gsk3b (glycogen synthase kinase 3 beta) conditional mutant, a modified rapamycin-binding domain derived from the mouse Mtor gene, flagged with a hemagglutinin tag (FRB*HA), takes the place of the endogenous stop codon, destabilizing the protein product. Homozygous mice die shortly after birth. The cause of death is not formally known, but mice are born with a cleft palate, sternal fusion defects, and they fail to nurse. Transcript levels and expression patterns reflect those of wildtype mice, but protein levels are are greatly reduced. Heterozygous mice are viable and appear normal.

Administration of rapamycin or a nontoxic, non-teratogenic, membrane-permeable rapamycin derivative (C20-methallylrapamycin; C20-MaRap), can re-establish stability and functional kinase activity of the protein as it binds to the ubiquitously expressed FKBP12 protein. C20-MaRap-mediated stabilization can be rapidly reversed by the addition of an FKBP12 binding competitor.

Gsk3b is a serine-threonine kinase involved in a range of biological processes including neurodevelopment, insulin-dependent glycogen synthesis, cellular proliferation and differentiation. This strain may be useful in studies of diabetes, inflammation, cardiovascular disease, cancer, neurodegenerative disease, and bipolar disorder.

Development

A loxP-flanked neomycin resistance cassette was cloned into the last intron of the targeted gene, and an FRB*HA sequence was inserted in place of the normal stop codon. FRB*HA encodes a K2095P, T2098L, W2101F mutant form of the FKBP-rapamycin binding (Frb) domain of Mtor (mechanistic target of rapamycin (serine/threonine kinase); FRAP) fused to hemagglutinin. The targeting vector was electroporated into TC1 129S6/SvEvTac-derived embryonic stem (ES) cells. The neomycin cassette was deleted by crossing resultant animals with mice expressing Cre recombinase from the β actin promoter (genetic background unknown). This strain was maintained on a mixed background incorporating 129S1/SvImJ and C57BL/6 by the donating laboratory. CD1 crosses are also mentioned in the primary reference.

Allele

Symbol: Gsk3b^tm1Grc
Name: targeted mutation 1, Gerald R Crabtree
MGI accession ID: MGI:3701999
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Gsk3b
Marker name: glycogen synthase kinase 3 beta
Chromosome: 16
Strain of origin: 129S6/SvEvTac
Molecular note: A mutant FRB tag, composed of an 89 amino acids with three point mutations (K2095P, T2098L, and W2101F), was targeted into the C terminus of the locus. A floxed neo cassette was inserted into the last intron and an FRB*HA sequence placed immediately before the stop codon. The neo was subsequently removed via crossing with mice expressing cre recombinase in the germline. Presence of this mutant FRB tag destabilizes the protein resulting in decrease or loss of expression. Addition of rapamycin or a rapamycin analogue stabilizes the protein and restores expression.