Inducible cell labeling and lineage tracking during fracture repair

Abstract

Mouse models incorporating inducible Cre-ER T2 /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 (Ai9 9 Col2a1-creER T2 ) to track contribution of chondrogenic progenitors during bone regeneration in a closed fracture model and Ai9 iUBC (Ai9 9 UBC-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.

Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing

“…A), an optimized tamoxifen‐responsive estrogen receptor (ERT) , to enable the temporal control of Cre expression driven by a tissue specific gene as well as to avoid Cre leakage. The Cre‐ERT2 is basally expressed in the cytoplasm of cells, but in the presence of 4‐hydroxytamoxifen (4‐OHT), it translocates to the nucleus to induce recombination . We knocked the CreERT2 construct into the endogenous locus of POU5F1 via CRISPR/Cas9 mediated homologous recombination, similar to the constitutive Cre recombinase (Fig.…”

Inducible transgenic expression of tripeptidyl peptidase 1 in a mouse model of late-infantile neuronal ceroid lipofuscinosis

“…First, with two of the tamoxifen-inducible cre-driver transgenes (Tg UBC-cre/ERT2 and Tg Thy1-cre/ERT2 ), there was constitutive expression of cre/ERT2 in the absence of tamoxifen treatment in brain. A number of studies have reported basal levels of cre recombination to variable degrees with the Tg UBC-cre/ERT2 strain and other cre/ERT2 drivers [ 20 – 22 ]. In the most recent [ 20 ], widespread basal cre-recombination driven by Tg UBC-cre/ERT2 was detected in various tissues including kidney, liver, heart and lung.…”

Lineage tracking of mesenchymal and endothelial progenitors in BMP-induced bone formation

“…Notably, the study by Wosczyna (14) also used a more endothelial specific VE-Cadherin cre reporter system, and this showed no co-staining with bone or cartilage. This highlights a challenge associated with lineage tracking studies, where the non-specificity and/or leakiness of Cre mouse models can lead to confounding results (35). Thus caution must be made with the interpretation of all tracking studies.…”