Characterization of a novel murine Sost ERT2 Cre model targeting osteocytes

Delphine B Maurel,Julian A Vallejo,Mark L Johnson,Michael J Wacker,Sarah L Dallas,Tsutomu Matsumoto,Marie A Harris,Marco Brotto,Stephen E Harris,Yukiko Kitase,Lynda F Bonewald

doi:10.1038/s41413-018-0037-4

Abstract

Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology, function and disease. While numerous Cre models exist to target gene recombination in osteoblasts and osteoclasts, few target osteocytes specifically, particularly mature osteocytes. Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5’ and 3’ regions of the Sost gene (Sost ERT2 Cre). Four founder lines were crossed with the Ai9 Cre reporter mice. One founder line showed high and specific activity in mature osteocytes. Bones and organs were imaged and fluorescent signal quantitated. While no activity was observed in 2 day old pups, by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or ‘leaky’ with age. The percentage of positive osteocytes increased following tamoxifen injection, especially in males, with 43% to 95% positive cells compared to 19% to 32% in females. No signal was observed in any bone surface cell, bone marrow, nor in muscle with or without tamoxifen injection. No spontaneous signal was observed in any other organ. However, with tamoxifen injection, a few positive cells were observed in kidney, eye, lung, heart and brain. All other organs, 28 in total, were negative with tamoxifen injection. However, with age, a muscle phenotype was apparent in the Sost-ERT2 Cre mice. Therefore, although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes, the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.

Highlights

The osteocyte was thought to be a passive cell, but in the last decade it has been shown to have many functions, such as regulation of bone formation and resorption, generation of endocrine factors that target kidney and muscle and mechanotransduction
As there is a critical need for additional osteocyte selective Cre models, we focused on creating a new Cre ERT2 disease sclerosteosis, where family members had massive bone model, driven by the Sost promoter
As there is a critical need for additional osteocyte selective Cre models, we generated a new Cre ERT2 model, driven by the Sost promoter

Summary

Introduction

The osteocyte was thought to be a passive cell, but in the last decade it has been shown to have many functions, such as regulation of bone formation and resorption, generation of endocrine factors that target kidney and muscle and mechanotransduction (reviewed in[1]). Reporter expression was mainly observed in osteocytes with low expression in osteoblasts. When this mouse was crossed with the Ai9 mouse in which the recombination event causes activation of tdTomato fluorescent protein (Jackson Labs), fluorescence was detected in several other tissues including muscle, brain, kidney, and the majority of osteoblasts on the bone surface.[9,10] Gorski and colleagues found that the Cre recombination in muscle was dependent on the animal.[14] Unintended targeting of Dmp1-Cre showed an effect on the gastrointestinal mesenchyme.[15] It is not clear why this variability in Cre activation occurs but it may be due to mouse strain and/or spontaneous activation during various stages of development

Objectives

Methods

Results

Conclusion