Searchable abstracts of presentations at key conferences on calcified tissues
Bone Abstracts (2016) 5 P294 | DOI: 10.1530/boneabs.5.P294

ECTS2016 Poster Presentations Osteoporosis: evaluation and imaging (39 abstracts)

Establishing an LRP5 mutant zebrafish (D. Rerio) model of bone acquisition

Chen Shochat , Ram Harari & David Karasik


Bar Ilan University, Zefat, Israel.

Aim & background: Evidence from genetic studies of osteoporosis and fracture risk indicate that bone mineral density (BMD) is genetically controlled. The low-density-lipoprotein-related receptor 5 (lrp5) gene is a known co-receptor in the canonical Wnt bone formation regulatory pathway. Although mutations found in this gene have been shown to regulate BMD in humans, and Lrp5 knockout mice had reduced bone mass, its role in the bone development/bone acquisition ambiguous. Their genetic similarity to humans, small size, and external development make zebrafish (ZF) a valuable model for vertebrate development.

Methods: We knocked out the lrp5 gene in ZF using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology. One-cell-stage ZF embryos were injected with CRISPR-Cas9 lrp5 guide RNA. Young adult-injected ZF were inbred to obtain F1 progeny. F1 siblings carrying mutations were then crossed to generate F2 progeny, and phenotype-genotype correlations were done using the F2 larvae. Mutation characterization was done by extracting DNA from lrp5 mutant ZF, PCR amplification and sequencing after cloning into pGEM-T vector. Phenotyping of mutated larval ZF was done by calcein staining of vertebrae detected by fluorescent microscope. Animal experiments were approved by the Bar Ilan University IACUC (no. 52-12-2012).

Results & conclusion: ZF injected with CRISPR-Cas9 lrp5 guide RNA were heterozygotes to lrp5 mutations. Mutations in F1 sibling were mainly small insertions or deletions of 3-7bp, some resulting in a null allele, and their F2 progeny were mainly compound heterozygotes to lrp5 mutations. The mutants did not differ in mineralized vertebrae number compared to WT at 8, 10 and 13 days post fertilization (dpf), which include first stages of endochondral ossification, although their body size was significantly lower at 13 dpf.

Understanding the role of lrp5 in bone acquisition will help identify novel therapeutic targets for preventing osteoporosis.

Volume 5

43rd Annual European Calcified Tissue Society Congress

Rome, Italy
14 May 2016 - 17 May 2016

European Calcified Tissue Society 

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