Increased trabecular bone and improved biomechanics in an osteocalcin null rat model created by CRISPR/Cas9 technology

Abstract

Osteocalcin, also known as bone γ-carboxyglutamate protein (Bglap), is expressed by osteoblasts and is commonly used as a clinical marker of bone turnover. A mouse model of osteocalcin deficiency has implicated osteocalcin as a mediator of changes to the skeleton, endocrine system, reproductive organs and central nervous system. However, differences between mouse and human osteocalcin at both the genome and protein levels have challenged the validity of extrapolating findings from the osteocalcin-deficient mouse model to human disease. The rat osteocalcin (Bglap) gene locus shares greater synteny with that of humans. To further examine the role of osteocalcin in disease, we created a rat model with complete loss of osteocalcin using the CRISPR/Cas9 system. Rat osteocalcin was modified by injection of CRISPR/Cas9 mRNA into the pronuclei of fertilized single cell Sprague-Dawley embryos, and animals were bred to homozygosity and compound heterozygosity for the mutant alleles. Dual-energy X-ray absorptiometry (DXA), glucose tolerance testing (GTT), insulin tolerance testing (ITT), microcomputed tomography (µCT), and a three-point break biomechanical assay were performed on the excised femurs at 5 months of age. Complete loss of osteocalcin resulted in bones with significantly increased trabecular thickness, density and volume. Cortical bone volume and density were not increased in null animals. The bones had improved functional quality as evidenced by an increase in failure load during the biomechanical stress assay. Differences in glucose homeostasis were observed between groups, but there were no differences in body weight or composition. This rat model of complete loss of osteocalcin provides a platform for further understanding the role of osteocalcin in disease, and it is a novel model of increased bone formation with potential utility in osteoporosis and osteoarthritis research.

An Osteocalcin-deficient mouse strain without endocrine abnormalities

“…Rats, like humans, have only a single Bglap locus. Bglap knockout rats do not have elevated glucose levels, insulin resistance, or decreased male fertility [23]. To date, genetic studies in humans have also not identified a role for OCN in these aspects of physiology.…”

A RATional choice for translational research?

“…Traditional quantitative trait locus studies, with the need for lengthy fine-mapping and testing of candidate genes, continue to give valuable insights (for example Zhao et al, 2015; Fernandez-Duenas et al, 2015; Wang et al, 2016, and reviewed in Yau and Holmdahl, 2016), but, as with research using other model organisms, these approaches are being replaced by gene targeting using CRISPR/Cas9 and other techniques, as exemplified by the GERRC programme () and the osteocalcin study in this issue (Lambert et al, 2016). These programmes use gene targeting to knock out genes implicated in the aetiology of common human diseases via genome-wide association studies (GWAS) or other patient-based studies, and allow analysis of gene function that will give insights into disease pathogenesis.…”

“…Because the osteocalcin locus has undergone triplication in the mouse genome, the rat, which, like humans, has a single osteocalcin gene, has the potential to be more relevant for studies of human osteocalcin gene variation. Osteocalcin knockout rats demonstrated increased trabecular thickness and improved functional quality under biomechanical stress, suggesting new therapeutic approaches for osteoporosis and osteoarthritis (Lambert et al, 2016). …”

NF1deficiency correlates with estrogen receptor signaling and diminished survival in breast cancer

“…as have been observed in other studies. 34,35 To verify the presence of the smaller indels and the larger in-frame deletions in each animal, we employed a three-step process of HMA, restriction digest, and Sanger sequencing ( Figure 1D-E, Supplementary Table 1-2). The majority of smaller indels 10/19 (52.6%) were detected in the 5' CRISPR target region of G0 animals.…”