B6.129P2-Fbxw8^{Gt(RRT057)Byg}/JdecJ

Stock number: 018058
Research areas: Developmental Biology Research, Research Tools

Description

This gene trap mutation abolishes endogenous Fbxw8 gene function while expressing a β-geo reporter fusion protein from the disrupted locus. These Fbxw8^{Gt(pGTOLxf)1Jdec} mice may be useful as a lacZ reporter for Fbxw8 expression or as a knockout model for studying the ubiquitin-proteasome pathway, and early embryonic growth and development.

Detailed description

The FBXW8 protein is a component of cullin-RING ligase (SKP1-cullin-F box protein complex) that is part of the ubiquitin-proteasome pathway of protein degradation. These mice carry a mutation of the Fbxw8, F-box and WD-40 domain protein 8, gene. Heterozygous mice are viable and fertile. Homozygous mice have a perinatal lethal phenotype with 70% dying soon after birth. Homozygous embryos are viable until late gestation. Gene product (protein) is almost undetectable by Western blot analysis of MEFs from homozygotes. Low levels of gene product (mRNA), 0.8% of levels seen in wildtype controls, are detected in MEFs isolated from homozygotes, as analyzed by real time PCR. At E12.5, homozygotes are smaller in size than controls, and become progressively smaller. By E18.5, the size reduction of homozygous embryos and placentas is significant, 67% of littermate size and 60 to 67% of wildtype placenta size, respectively. Homozygous embryos, aged E18.5, display impaired respiration and quickly become cyanotic. Histological analysis of lungs from homozygous E18.5 embryos reveals diminished alveolar space. Mutant mice exhibit a thin and disorganized placental spongiotrophoblast layer. Homozygotes that survive postnatally are smaller in size than wildtype littermates and exhibit a slower growth rate. Homozygotes, aged 3 months, exhibit smaller organ weight for liver, spleen, kidney, lungs and heart, but have brains similar in size to wildtype mice. Strong β-galactosidase expression is detected in skeletal muscle, developing bone epiphyses, bronchial epithelia of homozygous E14.5 and E18.5 embryos. Weaker β-galactosidase expression is detected in smooth muscle, kidneys, eyes, skin, and brain. β-galactosidase expression is also detected in the placenta. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.

Development

A "gene trap" targeting vector was used replace endogenous F-box and WD-40 domain protein 8 (Fbxw8) gene function with a β-galactosidase (β-geo) fusion protein in 129P2/OlaHsd-derived embryonic stem (ES) cell line, RRT057. The gene trap contained a β-geo fusion protein inserted into intron 3, 2.6 kb downstream of the exon 3, of the Fbxw8 gene. These ES cells were injected into recipient blastocysts, and chimeric males were bred with C57BL/6 mice. The mice were subsequently backcrossed to C57BL/6 for 10 generations. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

Allele

Symbol: Fbxw8^{Gt(RRT057)Byg}
Name: gene trap RRT057, BayGenomics
MGI accession ID: MGI:3526146
Generation method: Gene trapped
Attributes: Reporter; Hypomorph
Marker symbol: Fbxw8
Marker name: F-box and WD-40 domain protein 8
Chromosome: 5
Strain of origin: 129P2/OlaHsd
Expressed genes: Bgeo,fusion of beta-galactosidase and neomycin phosphotransferase genes,E. coli
Site of expression: Strong lacZ expression is detected in skeletal muscle, developing bone epiphyses, bronchial epithelia of homozygous E14.5 and E18.5 embryos. Weaker expression is detected in smooth muscle, kidneys, eyes, skin, and brain.
Molecular note: A genetrap vector containing a beta-gal reporter and a neomycin selection cassette inserted into intron 3 of the gene locus, 2.6 kb downstream of the exon 3. Western analysis on homozygous embryonic fibroblasts indicated protein expression was at 0.8% of the level in wild-type embryonic fibroblasts. Beta gal expression was detected in numerous tissues of homozygotes including cartilage, skeletal muscle, bronchiolar epithelium, and placenta.