Generation of the VCP^R155H/+ knock-in mice.

<p>(A) Schematic drawing of the R155H targeting strategy of the knock-in (KI) allele. The top line shows the knock-in (KI) allele and the below line depicts the wild-type (WT) allele. The localizations of exons 1 through 5 are numbered. Localizations of the 5′ (7.9 kb) and 3′ (2.1 kb) targeting sequences are indicated by dashed lines. Neomycin-cassette is marked by Neo, flanked by FRT sites and the restriction enzyme sites are indicated as follows: K = Kpn I, Bg = Bgl II, R = EcoR I, H = Hind III. Black arrow heads flanking Neo cassette show LoxP sites and white arrow heads show FRT sites. Black arrows flanking exon 5 indicate the locations of primers used in genotyping. (B) PCR genotyping of isolated from mouse tail DNA and digested by Msp I produces 274 bp and 362 bp in wild-type, while mutant allele gives three fragments of 636 bp, 274 bp and 362 bp. (C) RT-PCR-Msp I digestion analysis of mRNA isolated from mouse quadriceps shows similar expression levels. Wild-type allele produces 700 bp and 282 bp fragments, and mutant allele produces a 982 bp fragment. Fragment sizes are shown on the right and genotypes above. (D) VCP expression by western immunoblot in tissues: quadriceps, brain, heart and liver in wild type compared with knock-in mouse model shows similar expression levels. (E) VCP expression by Western immunoblot in quadriceps from wild type compared with knock-in mouse model. Beta actin was used as a loading control for these western blot analyses.