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

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.