title: 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.
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Lambert
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Challa
authors.affiliation: Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Aidi
authors.initials: A
authors.lastname: Niu
authors.affiliation: Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Janusz
authors.initials: J
authors.lastname: Tucholski
authors.affiliation: Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Johnson
authors.affiliation: Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Tim R
authors.initials: TR
authors.lastname: Nagy
authors.affiliation: Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Alan W
authors.initials: AW
authors.lastname: Eberhardt
authors.affiliation: Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Patrick N
authors.initials: PN
authors.lastname: Estep
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kesterson
authors.affiliation: Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA Department of Surgery, Birmingham VA Medical Center, Birmingham, AL 35233, USA jgrams@uab.edu.
authors.forename: Jayleen M
authors.identifier: 0000-0002-6510-6995
authors.initials: JM
authors.lastname: Grams
mesh_terms: D015502:Absorptiometry, Photon; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001696:Biomechanical Phenomena; D001823:Body Composition; D064113:CRISPR-Cas Systems / Q000235:genetics*; D000071556:Cancellous Bone / Q000000981:diagnostic imaging / Q000502:physiology*; D005269:Femur / Q000000981:diagnostic imaging / Q000502:physiology; D018703:Founder Effect; D005821:Genetic Techniques; D005951:Glucose Tolerance Test; D054643:INDEL Mutation / Q000235:genetics; D007328:Insulin / Q000378:metabolism; D008297:Male; D023421:Models, Animal; D015675:Osteocalcin / Q000737:chemistry / Q000172:deficiency* / Q000378:metabolism; D051381:Rats; D013045:Species Specificity; D055114:X-Ray Microtomography
chemical_list: D007328:Insulin; D015675:Osteocalcin
keywords: Bone strength; Bone structure; Genetic animal models; Osteocalcin; Osteocalcin knockout