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

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 <i>Tg(Dmp1-Cre)</i> and <i>Tg(Dmp1-CreERT2)</i> lines, while widely used and helpful, utilize transgenic approaches with transgene expression driven by the <i>Dmp1</i> 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-<i>Dmp1^em1(CreERT2)</i> and <i>Dmp1^em2(ZsGreen)</i> -via CRISPR-Cas9 mediated "knock-in" approaches to insert an internal ribosome entry site (<i>IRES</i>)<i>-CreERT2</i> or <i>IRES-ZsGreen</i> cassette into the <i>Dmp1</i> locus, allowing <i>CreER^T2</i> or <i>ZsGreen</i> expression under the genetic control of the endogenous <i>Dmp1</i> locus while preserving <i>Dmp1</i> gene expression. We show that the <i>Dmp1^em1(CreERT2)</i> enabled highly efficient tamoxifen-inducible recombination (>90% in cortical and trabecular osteocytes) with minimal off-target expression in non-bone tissues. The <i>Dmp1^em2(ZsGreen)</i> line showed robust, fixation-, and decalcification-resistant green fluorescence in osteocytes from birth through adulthood, faithfully reflecting endogenous <i>Dmp1</i> expression. Lineage tracing using the <i>Dmp1^em1(CreERT2)</i> 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.