New knock-in mouse lines Dmp1em1(CreERT2) and Dmp1em2(ZsGreen) 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 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-Dmp1em1(CreERT2) and Dmp1em2(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 CreERT2 or ZsGreen expression under the genetic control of the endogenous Dmp1 locus while preserving Dmp1 gene expression. We show that the Dmp1em1(CreERT2) enabled highly efficient tamoxifen-inducible recombination (>90% in cortical and trabecular osteocytes) with minimal off-target expression 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 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.