A GCaMP reporter mouse with chondrocyte specific expression of a green fluorescent calcium indicator

IF 3.5 2区 医学 Q2 ENDOCRINOLOGY & METABOLISM Bone Pub Date : 2024-08-13 DOI:10.1016/j.bone.2024.117234
Sotirios A. Tsadaris , David E. Komatsu , Vladimir Grubisic , Raddy L. Ramos , Michael Hadjiargyrou
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Abstract

One of the major processes occurring during the healing of a fractured long bone is chondrogenesis, leading to the formation of the soft callus, which subsequently undergoes endochondral ossification and ultimately bridges the fracture site. Thus, understanding the molecular mechanisms of chondrogenesis can enhance our knowledge of the fracture repair process. One such molecular process is calciun (Ca++) signaling, which is known to play a critical role in the development and regeneration of multiple tissues, including bone, in response to external stimuli. Despite the existence of various mouse models for studying Ca++ signaling, none of them were designed to specifically examine the skeletal system or the various musculoskeletal cell types. As such, we generated a genetically engineered mouse model that is specific to cartilage (crossed with Col2a1 Cre mice) to study chondrocytes. Herein, we report on the characterization of this transgenic mouse line using conditional expression of GCaMP6f, a Ca++-indicator protein. Specifically, this mouse line exhibits increased GCaMP6f fluorescence following Ca++ binding in chondrocytes. Using this model, we show real-time Ca++ signaling in embryos, newborn and adult mice, as well as in fracture calluses. Further, robust expression of GCaMP6f in chondrocytes can be easily detected in embryos, neonates, adults, and fracture callus tissue sections. Finally, we also report on Ca++ signaling pathway gene expression, as well as real-time Ca++ transient measurements in fracture callus chondrocytes. Taken together, these mice provide a new experimental tool to study chondrocyte-specific Ca++ signaling during skeletal development and regeneration, as well as various in vitro perturbations.

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特异性表达软骨细胞绿色荧光钙指示剂的 GCaMP 报告小鼠
软骨生成是长骨骨折愈合过程中发生的主要过程之一,它导致软茧的形成,软茧随后发生软骨内骨化,最终在骨折部位形成桥接。因此,了解软骨生成的分子机制可以增进我们对骨折修复过程的了解。众所周知,钙离子(Ca++)信号传导在包括骨骼在内的多种组织的发育和再生过程中对外界刺激起着至关重要的作用。尽管存在各种研究 Ca++ 信号传导的小鼠模型,但它们都不是专门为研究骨骼系统或各种肌肉骨骼细胞类型而设计的。因此,我们生成了一种专门针对软骨的基因工程小鼠模型(与 Col2a1 Cre 小鼠杂交)来研究软骨细胞。在此,我们报告了利用条件性表达钙离子指示蛋白 GCaMP6f 的转基因小鼠品系的特征。具体来说,该小鼠品系在软骨细胞与 Ca++ 结合后显示出更强的 GCaMP6f 荧光。利用这一模型,我们在胚胎、新生小鼠、成年小鼠以及骨折胼胝体中显示了实时 Ca++ 信号传导。此外,在胚胎、新生儿、成年小鼠和骨折胼胝体组织切片中都能轻松检测到软骨细胞中 GCaMP6f 的强表达。最后,我们还报告了Ca++信号通路基因的表达,以及骨折胼胝体软骨细胞中Ca++瞬态的实时测量。总之,这些小鼠为研究骨骼发育和再生过程中软骨细胞特异性 Ca++ 信号传导以及各种体外扰动提供了一种新的实验工具。
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来源期刊
Bone
Bone 医学-内分泌学与代谢
CiteScore
8.90
自引率
4.90%
发文量
264
审稿时长
30 days
期刊介绍: BONE is an interdisciplinary forum for the rapid publication of original articles and reviews on basic, translational, and clinical aspects of bone and mineral metabolism. The Journal also encourages submissions related to interactions of bone with other organ systems, including cartilage, endocrine, muscle, fat, neural, vascular, gastrointestinal, hematopoietic, and immune systems. Particular attention is placed on the application of experimental studies to clinical practice.
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