Project Summary Tendon and ligament injuries are widespread and affect young active to aging individuals. Once injured, tendons undergo slow and limited healing and never regain their original properties. This can result in limited mobility, pain associated with movements, and a high propensity for reinjury. By gaining a mechanistic understanding of the cell types and pathways regulating tendon cells during their formation, homeostasis, and repair, we hope to identify new potential ways to treat tendon injuries and improve tendon healing in patients. Studies using mouse genetic approaches have accelerated tendon research, identifying new tendon cell types and pathways that regulate formation and healing. Constitutive and inducible tendon recombinase lines, such as Scx-Cre and Scx-CreERt2, have been instrumental in allowing more tissue-specific gene loss of function analysis. However, there remain limitations in our ability to target specific subsets of tendon cells or to target tendons in distinct anatomical regions. Scx-Cre/Scx-CreERt2 as well as other Cre/CreERt2 lines used to label tendons and ligaments are not specific to these tissues, which can confound interpretation of cell lineage and functional studies. More specific tools are needed in order to advance future research and enable studies dissecting specific tendon cell subsets or anatomical regions. In the neuroscience field, intersectional genetic tools have been used for more than a decade to target developmentally and molecularly distinct cell networks and dissect their function. Therefore, we propose to generate new alleles that would enable intersectional genetic approaches in the tendon and could be applicable to other musculoskeletal tissues. First, we will generate constitutive and inducible Flp alleles that can be used with a large number of existing Cre/CreERt2 lines and Flp/Cre indicator alleles for intersectional cell labeling. This would allow specific targeting of subsets of tendon cells as well as targeting tendons in distinct anatomic locations. In addition, we propose the generation of two Flp/Cre dual recombinase responsive alleles for performing functional analysis of the intersecting cell populations via cell ablation or expression of rtTA, which can be used for functional perturbations when combined with already available TetO lines. Proper efficiency and specificity of all alleles will be validated using existing indicator or Cre/Flp lines. Together, the generation of these alleles will permit unprecedented targeting of specific tendon cell populations for lineage tracing and functional analysis. Their use by the musculoskeletal community would enable mechanistic studies and advance research with the goal of impacting the development of future tendon therapies.