An osteocalcin-deficient mouse strain without endocrine abnormalities
An osteocalcin-deficient mouse strain without endocrine abnormalities
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DOI：
10.1371/journal.pgen.1008361
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发表时间：
2020-05-01
期刊：
影响因子：
4.5
通讯作者：
Williams, Bart O.
中科院分区：
文献类型：
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作者：
Diegel, Cassandra R.;Hann, Steven;Williams, Bart O.
骨钙素（Osteocalcin， OCN）是骨基质中最丰富的非胶原蛋白，是一种骨源性内分泌激素，对生理的许多方面有广泛的影响，包括葡萄糖代谢和男性生育能力。许多这些观察结果是使用ocn缺陷小鼠等位基因（Osc(-)）进行的，其中小鼠的2个ocn编码基因Bglap和Bglap2在ES细胞中通过同源重组被删除。在这里，我们描述了具有新的Bglap和Bglap2双敲除（dko）等位基因（Bglap/2(p.p pro25fs17ter)）的小鼠，该等位基因是通过CRISPR/ cas9介导的基因编辑产生的。这种新等位基因纯合的小鼠不表达全长Bglap或Bglap2 mRNA，血清中没有免疫检测到的OCN。在这些纯合子敲除动物的皮质骨中，FTIR成像发现与野生型幼崽相比，皮质骨中的胶原成熟度和碳酸盐与磷酸盐比率发生了变化。然而，mu CT和三点弯曲试验没有发现野生型幼崽在骨量和强度方面的差异。与先前报道的具有Osc（-）等位基因的ocn缺陷小鼠相比，具有Bglap/2（pPro25fs17Ter）等位基因的ocn缺陷小鼠的血清葡萄糖水平和雄性生育能力与野生型幼崽没有显著差异。我们无法解释携带这种新的基因敲除等位基因的小鼠没有内分泌影响。可能的解释包括每个突变等位基因对邻近基因转录的影响，或遗传背景和环境的差异。因此，我们的发现可以被其他感兴趣的研究者证实和扩展，我们将这种新的Bglap和Bglap2双敲除菌株捐赠给杰克逊实验室进行学术分发。作者总结：制造和维持骨的细胞表达局部或系统功能的蛋白质。前者蛋白质，如1型胶原蛋白，影响骨骼的材料特性，而后者，如成纤维细胞生长因子23，使骨骼能够与其他器官系统进行通信。影响大多数骨细胞表达蛋白功能的突变导致的疾病在人类和其他哺乳动物（如小鼠）中具有类似的特征，例如，1型胶原蛋白突变导致脆性骨病，成纤维细胞生长因子23突变导致低磷血症佝偻病。我们的研究重点是另一种骨细胞表达蛋白，骨钙素，它被认为是局部影响骨强度和系统的激素。通过骨钙素敲除小鼠的研究，其他研究者提出骨钙素在调节血糖、男性生育能力、肌肉质量、大脑发育、行为和认知方面的内分泌作用。因此，我们决定产生一种新的骨钙素敲除小鼠菌株，也可用于研究这些非骨骼效应。令我们惊讶的是，我们创造的骨钙素敲除小鼠在我们检查的三种表型上与野生型小鼠没有显著差异：骨骼强度、血糖和雄性生育能力。我们的数据与骨钙素敲除大鼠的结果一致，但与原始骨钙素敲除小鼠的数据不一致。因为我们不知道为什么我们的新菌株不能重现先前在另一个敲除小鼠染色中报告的表型，我们已经将我们的小鼠捐赠给公共储存库，这样它们就可以很容易地在其他学术实验室中获得和研究。
Osteocalcin (OCN), the most abundant noncollagenous 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/2(p.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 serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, mu CT 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, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/2(pPro25fs17Ter) 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. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or 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 Laboratories for academic distribution.Author summaryCells that make and maintain bone express proteins that function either locally or systemically. The former proteins, such as type 1 collagen, affect the material properties of the skeleton, while the latter, 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 mutations in fibroblast growth factor 23. Our study focuses on another bone-cell-expressed protein, osteocalcin, which has been suggested to function locally to affect bone strength and systemically as a hormone. Studies using osteocalcin knockout mice led other investigators to suggest endocrine roles for osteocalcin in regulating blood glucose, 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 nonskeletal effects. To our surprise, the osteocalcin knockout mice we created did not significantly differ from wild-type mice for the three phenotypes we examined: bone strength, blood glucose, and male fertility. Our data are consistent with findings from osteocalcin knockout rats but are inconsistent with data from the original osteocalcin knockout mice. Because we do not know why our new strain fails to recapitulate the 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.