Inducible cell labeling and lineage tracking during fracture repair

摘要

Mouse models incorporating inducible Cre-ERT2/LoxP recombination coupled with sensitive fluorescent reporter lines are being increasingly used to track cell lineages in vivo. In this study we use two inducible reporter strains, Ai9(iCol2a1) (Ai9xCol2a1-creER(T2)) to track contribution of chondrogenic progenitors during bone regeneration in a closed fracture model and Ai9(iUBC) (Ai9xUBC-creER(T2)) to examine methods for inducing localized recombination. By comparing with Ai9 littermate controls as well as inducible reporter mice not dosed with tamoxifen, we revealed significant leakiness of the CreER(T2) system, particularly in the bone marrow of both lines. These studies highlight the challenges associated with highly sensitive reporters that may be activated without induction in tissues where the CreER(T2) fusion is expressed. Examination of the growth plate in the Ai9(iCol2a1) strain showed cells of the osteochondral lineage (cell co-staining with chondrocyte and osteoblast markers) labeled with the tdTom reporter. However, no such labeling was noted in healing fractures of Ai9(iCol2a1) mice. Attempts to label a single limb using intramuscular injection of 4-hydroxytamoxifen in the Ai9(iUBC) strain resulted in complete labeling of the entire animal, comparable to intraperitoneal injection. While a challenge to interpret, these data are nonetheless informative regarding the limitations of these inducible reporter models, and justify caution and expansive controls in future studies using such models.