New knock in mouse lines Dmp1em1(CreERT2) and Dmp1em2(ZsGreen) enable precise osteocyte-specific targeting and visualization

Abstract

In adult mammals, bone is a dynamic organ with both structural and endocrine functions, mainly mediated by osteocytes, the most abundant bone cells, and the principal source of bone-derived hormones such as fibroblast growth factor 23 (FGF23) and sclerostin (SOST). The widely used 10-kb promoter-driven transgenic Tg(Dmp1-Cre) and Tg(Dmp1-CreERT2) lines, while widely used and helpful, utilize transgenic approaches with transgene expression driven by the Dmp1 promoter. The transgene expression suffers from positional effects with off-target expression in muscle, brain, and other tissues, limiting their applications, causing a major technical gap in the investigation of osteocyte-specific functions. Here, we generated two novel mouse lines, Dmp1em1(CreERT2) and Dmp1em2(ZsGreen), via CRISPR-Cas9-mediated knock-in approaches that allow CreERT2 or ZsGreen expression under the genetic control of the endogenous Dmp1 locus by inserting an IRES–CreERT2 or IRES–ZsGreen cassette into the Dmp1 locus, preserving the Dmp1 gene expression. We showed that the Dmp1em1(CreERT2) enabled highly efficient tamoxifen-inducible recombination (>90% in cortical and trabecular osteocytes) with minimal off-target labeling in non-bone tissues. The Dmp1em2(ZsGreen) line showed robust, fixation- and decalcification-resistant green fluorescence in osteocytes from birth through adulthood, faithfully reflecting endogenous Dmp1 expression. Lineage tracing using the Dmp1em1(CreERT2) line further revealed that the cortical osteocytes are long-lived, whereas trabecular osteocytes undergo continuous renewal, uncovering compartment-specific differences in osteocyte lifespan. By eliminating off-target recombination or gene expression in skeletal muscle, brain, gastrointestinal tract, and marrow compartments, the two mouse lines offer powerful tools for rigorous studies in osteocyte biology, mechanotransduction, and bone regulation of systemic physiology that impact health, aging, and diseases, while minimizing complications due to off-target transgene expression.