New knock-in mouse lines Dmp1^em1(CreERT2) and Dmp1^em2(ZsGreen) enable precise osteocyte-specific targeting and...

Bone in adult mammals plays critical structural and endocrine functions, both largely rely on osteocytes-the most abundant bone cells and principal source of bone-derived hormones, such as fibroblast growth factor 23 and sclerostin. 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 2 novel mouse lines-Dmp1^em1(CreERT2) and Dmp1^em2(ZsGreen) -via CRISPR-Cas9 mediated "knock-in" approaches to insert an internal ribosome entry site (IRES)-CreERT2 or IRES-ZsGreen cassette into the Dmp1 locus, allowing CreER^T2 or ZsGreen expression under the genetic control of the endogenous Dmp1 locus while preserving Dmp1 gene expression. We show that the Dmp1^em1(CreERT2) enabled highly efficient tamoxifen-inducible recombination (>90% in cortical and trabecular osteocytes) with minimal off-target expression in non-bone tissues. The Dmp1^em2(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 Dmp1^em1(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 2 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.