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Bone Abstracts (2019) 7 OC19 | DOI: 10.1530/boneabs.7.OC19

1University of Pavia, Pavia, Italy; 2University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 3Ghent University, Ghent, Belgium.

Objectives: Osteogenesis imperfecta (OI) is a heritable disorder characterized by bone deformity and skeletal fragility. Cartilage associated protein (CRTAP), proline 3-Hydroxylase 1 (P3H1) and Cyclophylin B (PPIB) are components of the endoplasmic reticulum (ER)-resident complex involved in the 3-hydroxylation of specific proline residues in collagen type I α chains. Mutations in these proteins are responsible for recessive OI type VII, VIII and IX, respectively. Murine models for these diseases exist, but our goal was to exploit the availability of zebrafish models to deeper understand the phenotype at early developmental stages and to favour drug screening reducing amount, timing and cost.

Methods: CHOPCHOP was used for guide RNA selection. pT7gRNA vector was used for gRNA subcloning and in vitro transcription and the pT3TS-nCas9n plasmid for Cas9 mRNA synthesis. Alizarin red staining, μCT and X-Rays were used for bone characterization. The growth rate of caudal fin was performed by measuring regrown tail after amputation. Collagen type I was characterized by SDS-PAGE. Collagen fibers were analysed by transmission electron microscopy (TEM).

Results: OI type VII and VIII knock out zebrafish models were generated using CRISPR Cas9 system. P3h1 and Crtap mutants are smaller than WT and show a delayed mineralization starting from the first weeks of life. Their phenotype is worsening with age, as observed by the severe skeletal deformities present during adulthood. Bone formation, evaluated on fin regeneration, is delayed in p3h1 mutants compared to WT. Collagen type I is overmodified in both mutants, as suggested by the broadened α bands observed in SDS-PAGE. TEM images show smaller collagen fibers diameters in mutants with respect to WT. An enlargement of ER cisternae is evident in mutants, suggesting the presence of ER stress due to collagen retention.

Conclusion: We proved the goodness of zebrafish model to reproduce the phenotype of recessive OI type VII and VIII. Since ER stress could be a potential therapeutic target, our goal will be to use our models for drug screening in order to pave the way to new pharmacological treatments.

Funding: Telethon [grant n. GGP13098] and the European Community, FP7, ‘Sybil’ project [grant n. 602300].

Disclosure: The authors declared no competing interests.

Volume 7

9th International Conference on Children's Bone Health


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