Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane

Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. developmental timing of muscle LY2886721 deletion supports a role for dystroglycan in muscle development or differentiation. Moreover, given that this conditional (11). mice exhibit muscle and brain phenotypes, loss of functional DG glycosylation, and reduced lifespan. Although this is a very useful model, the presence of severe brain abnormalities impairs mouse growth and movement and can hinder more in-depth studies of the muscle disease. To better understand disease mechanisms and characterize DG hypoglycosylation in dystroglycanopathies, we sought to develop a mouse model for conditional disruption of DG processing. We targeted the dystroglycanopathy gene disruption during muscle development at E8 causes a more severe phenotype than KO initiated at E17 in differentiating muscle and discovered that FKTN activity is necessary at or near DGs unusual exon 2 for conditional deletion of the start codon. Both null (Cre-recombined) and floxed (neo cassette removed) alleles were generated using Cre/LoxP or Flp/Frt germline recombination, respectively (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI63004DS1). Consistent with previous reports, we could not obtain complete KO mice, confirming embryonic lethality of germline disruption (Supplemental Figure 1D and refs. 17, 23). Surprisingly, the incidence of homozygosity for the floxed allele was also sub-Mendelian, indicating that some lethality is associated with floxed exon 2 even in the absence of Cre recombinase (Supplemental Figure 1D). mice that survived to birth were indistinguishable from wild-type littermates. Whole-animal Fktn disruption impairs DG glycosylation. To validate Cre recombination at the locus and confirm disruption of DG glycosylation in vivo, we used whole-animal tamoxifen-inducible (Tam-inducible) Cre mice (TgCre-Esr1; The Jackson Laboratory, no. 004682) to induce exon 2 deletion. TgCre-Esr1;mice (iKO) and littermates were administered Tam or vehicle (Veh) at 6 weeks of age. Tam-treated iKO (Tam iKO) mice LY2886721 showed no difference in body weight, forelimb grip strength, or open-field activity compared with littermates from 4 through 20 weeks of age (data not shown). In contrast, serum creatine kinase (CK) levels in Tam iKO mice began to rise at 14 weeks, and were significantly higher than in control mice at 16 and 20 weeks of age, indicating muscle damage (Figure ?(Figure1C1C and data not shown). Histological analysis of the iliopsoas muscle at 20 weeks confirmed the presence of dystrophic disease, with variation in fiber size, necrosis, and a significant increase in centrally nucleated fibers (Figure ?(Figure1,1, A and B), indicative of muscle regeneration. Figure 1 Inducible, whole-animal deletion causes dystrophic features with disruption of DG glycosylation. Staining for the DG glyco-epitope was nearly negative or patchy, while DG remained at the sarcolemma of Tam iKO mice, indicating a disruption in DG processing rather than abnormal protein expression (Figure ?(Figure1A).1A). This was confirmed by Western blotting with an antibody directed to the DG core protein; DG protein was present with a reduced molecular weight in Tam iKO muscle (Figure ?(Figure1D).1D). DG glycosylation was partially disrupted in some Veh iKO mice (Figure ?(Figure1D);1D); however, LY2886721 histological analysis of 2 Veh iKO mice in this group revealed no significant abnormalities (data not shown). We hypothesized that the reduced DG molecular weight in these Veh iKO mice was due to exposure to Tam while mice were cohoused with Tam littermates, so we performed additional experiments on Veh iKO Itga10 mice caged separately from Tam littermates for 7 days after the first Tam dose or iKO mice from cages receiving no treatment. LY2886721 DG glycosylation was normal in these uninduced iKO samples (Supplemental Figure 2A, 10 weeks after vehicle iKO [age 16 weeks]; and 20-week-old iKO; data not shown), confirming that untreated mice can be exposed to Tam from treated cagemates. To assess the time from induction of gene excision to loss of DG glycosylation, we Tam treated additional iKO mice and collected muscle tissues for analysis at various times. While a small amount of normal DG was detected in wheat germ agglutininCenriched (WGA-enriched) skeletal muscle of.