Targeted gene deletion specific to adult periosteum has not been realized due to poorly defined cell populations and the absence of specific markers. The live segmental bone graft transplantation approach allows us to indiscriminately analyze the residing progenitor cells via tracking donor cell fate and evaluating their contribution to healing. If combined with a TM inducible CreER mouse model, this approach could achieve an inducible gene targeting in early periosteal stem/progenitor cells. To obtain more efficient and ubiquitous deletion in periosteum, we choose to use R26CreER mice7
. To determine the efficiency of TM-induced Cre-mediated recombination, we crossed R26CreER with a LacZ reporter R26R mouse. The R26R mice consist of a loxP
flanked neo expression cassette upstream of the LacZ gene, which prevents transcriptional read-through of the LacZ gene. When crossed with a strain that expresses Cre recombinase, the stop sequence will be removed in Cre expressing cells thereby activating LacZ gene expression. As demonstrated in , strong LacZ staining was found in the tailbone of R26CreER; R26R mice following TM ip. injection (). In contrast, no LacZ positive staining was found in the control non-TM treated mice ().
Figure 1 A global Tamoxifen inducible model for targeted gene recombination. R26CreER;R26R mice were treated with TM three times at a dose of 1mg/day. X-Gal staining was performed in mouse tailbones. LacZ staining was observed in nearly all cells including chondrocytes, (more ...)
To determine whether a selective gene recombination in periosteal progenitor cells can be achieved via bone graft transplantation, R26CreER;R26R mice were treated with 1mg of TM for consecutive 3 days to induce global gene recombination. Following the treatment, the femoral bone graft from a RosaCreER;RosaR mouse was harvested and immediately implanted into a mid-diaphyseal defect created in non-TM treated RosaCreER littermates. schematically illustrates the approach as described. The grafted femoral samples were harvested on day 7 and processed for X-Gal staining. As shown in , large numbers of LacZ positive cells were found at the cortical bone junctions, similar to what we have previously observed in R26A transplantation6
. At the graft side, nearly 70% of mesenchymal cells and chondrocytes were stained as LacZ positive (), confirming the effective gene targeting in periosteal progenitor cells.
Figure 2 Tamoxifen-inducible CreER mediated gene targeting in early periosteal callus can be achieved via bone graft transplantation. Schematic illustration of the graft transplantation approach using R26CreER;R26R mice (A). Following TM treatment, bone grafts (more ...)
To further characterize the efficiency of TM induced recombination in repair tissue, a mid-diaphyseal femur fracture was created in R26CreER;R26R mouse. TM was administered for one (day1), two (day 1 and 3) and three times (day 1, 3 and 5) post-fracture at a dose of 1mg/mouse. All samples were harvested at day10 post-fracture. As demonstrated in , two ip. injections of TM were sufficient to induce recombination in nearly 70% of the reparative tissues in fracture callus (). And three-time injection of TM induced recombination in 80% of repair tissue in fracture callus (). Remarkably, we found that the percentage of LacZ positive cells was much higher in the callus than in its surrounding tissue or in growth plate, particularly at low dosing treatment ().
Figure 3 Efficient CreER-mediated gene recombination in the fracture repair tissues. A femoral fracture was created in R26CreER;R26R mice and TM was administered once (day1), twice (day 1 and 3) or three times (day 1,3 and 5) post-fracture at a dose of 1mg/day/mouse. (more ...)
In view of the fact that mesenchymal stem cells come from different sources, the global inducible model holds a better chance to induce recombination in various adult progenitor cell pools. The efficient recombination in early stem/progenitor cells further results in an efficient removal of the targeted gene from all progeny following cortical bone fracture or osteotomy. The presented data represent a novel approach to achieve efficient global gene targeting in adult repair tissues. If combined with bone graft transplantation, a selectively targeted gene deletion can be achieved only in periosteal progenitor cells and their progeny around bone graft. When incorporated with appropriate floxed mice, this approach can be used to examine the function of genes in controlling donor periosteal progenitor cell fate and cell dependent healing in adult repair. Another advantage of using a bone-grafting approach is that TM is administered to induce gene recombination in donor cells prior to transplantation. After transplantation, the mice will not receive any TM therefore completely eliminating its potential negative effects on bone graft healing.
Identifying essential signals for stem/progenitor dependent bone healing is of paramount importance. Our inability to treat patients with impaired bone healing is largely due to our limited understanding of the molecular signals or signaling that are essential for this process. The current study represent a novel approach among availalable technologies to examine the function of a single gene in adult bone and cartilage tissue repair. This approach is extremely useful in delineating the function of genes whose conventional deletion results lethality during development.