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An Osteocalcin-deficient mouse strain without endocrine abnormalities

Cassandra R. Diegel, Steven Hann, Ugur M. Ayturk, View ORCID ProfileJennifer C.W. Hu, Kyung-eun Lim, Casey J. Droscha, Zachary B. Madaj, Gabrielle E. Foxa, View ORCID ProfileIsaac Izaguirre, VARI Vivarium and Transgenics Core, Noorulain Paracha, View ORCID ProfileBohdan Pidhaynyy, View ORCID ProfileTerry L. Dowd, Alexander G. Robling, Matthew L. Warman, View ORCID ProfileBart O. Williams
doi: https://doi.org/10.1101/732800
Cassandra R. Diegel
1Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI 49503
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Steven Hann
4Orthopedic Research Labs, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, MA 02115
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Ugur M. Ayturk
4Orthopedic Research Labs, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, MA 02115
5Musculoskeletal Integrity Program, Hospital for Special Surgery Research Institute, New York, NY 10021
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Jennifer C.W. Hu
4Orthopedic Research Labs, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, MA 02115
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Kyung-eun Lim
6Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202
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Casey J. Droscha
1Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI 49503
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Zachary B. Madaj
2Bioinformatics and Biostatistics Core, Van Andel Research Institute, Grand Rapids, MI 49503
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Gabrielle E. Foxa
1Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI 49503
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Isaac Izaguirre
1Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI 49503
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3Vivarium and Transgenics Core, Van Andel Research Institute, Grand Rapids, MI 49503
Noorulain Paracha
8Department of Biology, Brooklyn College, Brooklyn, NY
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Bohdan Pidhaynyy
8Department of Biology, Brooklyn College, Brooklyn, NY
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Terry L. Dowd
7Department of Chemistry, Brooklyn College, Brooklyn, NY
9Ph.D. Program in Chemistry and Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, New York, NY 10016
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Alexander G. Robling
6Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202
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Matthew L. Warman
4Orthopedic Research Labs, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, MA 02115
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Bart O. Williams
1Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI 49503
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  • For correspondence: bart.williams@vai.org
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Abstract

Osteocalcin (OCN), the most abundant non-collagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc-) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their plasma. FTIR imaging of cortical and trabecular bone in these homozygous knockout animals finds alterations in the crystal size and maturity of the bone mineral, hydroxyapatite, compared to wild-type littermates; however, μCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc- allele, blood glucose levels and male fertility in the OCN-deficient mice with Bglap/2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Potential explanations include effects of each mutated allele on the transcription of neighboring genes, and differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double knockout strain to The Jackson Laboratory for academic distribution.

Author Summary Cells that make and maintain bone express proteins that function locally or systemically. The former proteins, such as type 1 collagen, affect the material properties of the skeleton while the latter proteins, such as fibroblast growth factor 23, enable the skeleton to communicate with other organ systems. Mutations that affect the functions of most bone cell expressed proteins cause diseases that have similar features in humans and other mammals, such as mice; for example, brittle bone diseases for type 1 collagen mutations and hypophosphatemic rickets for fibroblast growth factor 23 mutations.

Our study focuses on another bone cell expressed protein, osteocalcin, which has been suggested to function locally to affect bone strength and systemically as hormone. Studies using osteocalcin knockout mice led other investigators to suggest endocrine roles for osteocalcin in regulating blood glucose levels, male fertility, muscle mass, brain development, behavior and cognition. We therefore decided to generate a new strain of osteocalcin knockout mice that could also be used to investigate these non-skeletal effects.

To our surprise the osteocalcin knockout mice we created do not significantly differ from wild-type mice for the 3 phenotypes we examined: bone strength, blood glucose levels, and male fertility. Our data are consistent with findings from osteocalcin knockout rats, but inconsistent with data from the original osteocalcin knockout mice. Because we do not know why our new strain of osteocalcin knockout mice fails to recapitulate phenotypes previously reported for another knockout mouse stain, we have donated our mice to a public repository so that they can be easily obtained and studied in other academic laboratories.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted August 13, 2019.
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An Osteocalcin-deficient mouse strain without endocrine abnormalities
Cassandra R. Diegel, Steven Hann, Ugur M. Ayturk, Jennifer C.W. Hu, Kyung-eun Lim, Casey J. Droscha, Zachary B. Madaj, Gabrielle E. Foxa, Isaac Izaguirre, VARI Vivarium and Transgenics Core, Noorulain Paracha, Bohdan Pidhaynyy, Terry L. Dowd, Alexander G. Robling, Matthew L. Warman, Bart O. Williams
bioRxiv 732800; doi: https://doi.org/10.1101/732800
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An Osteocalcin-deficient mouse strain without endocrine abnormalities
Cassandra R. Diegel, Steven Hann, Ugur M. Ayturk, Jennifer C.W. Hu, Kyung-eun Lim, Casey J. Droscha, Zachary B. Madaj, Gabrielle E. Foxa, Isaac Izaguirre, VARI Vivarium and Transgenics Core, Noorulain Paracha, Bohdan Pidhaynyy, Terry L. Dowd, Alexander G. Robling, Matthew L. Warman, Bart O. Williams
bioRxiv 732800; doi: https://doi.org/10.1101/732800

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