Construction of the GAN-/- mouse. (A) Schematic representation of the disruption of the GAN gene. The targeted construct was designed to replace the endogenous exons 3-5 GAN locus with a neomycin-resistant gene (neo R). The diphteria toxin gene, placed at the 3' end of the targeting vector allowed positive selection for the homologous recombination at the GAN locus. Proper targeting produces an open reading frame of only 110 amino acids terminated by a premature stop codon in exon 6. The Δ GAN locus will only produce a Δ gigaxonin of 12 kDa, possibly unstable and entirely lacking the Kelch domain and a portion of the BTB domain. (B, C) Analysis of the genomic DNA from Wild Type (WT), heterozygous (Het) and GAN knock out mice (KO) by polymerase chain reaction (PCR) (B) and Southern blot after BglII digestion (C). The Embryonic Stem cell (ES) corresponds to the original GAN targeted clone. (D) Brain extracts (50 μg) from WT and GAN knock out mouse, and COS cell lysates (cosT; 0,5 μg) transfected with WT-gigaxonin were analysed by immunoblot using anti-Gigaxonin antibody.