Inducible cell labeling and lineage tracking during fracture repair
Inducible cell labeling and lineage tracking during fracture repair
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DOI：
10.1111/dgd.12184
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发表时间：
2015-01-01
影响因子：
2.5
通讯作者：
Schindeler, Aaron
中科院分区：
文献类型：
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作者：
Seime, Till;Kolind, Mille;Schindeler, Aaron
将可诱导的Cre-ERT2/LoxP重组与敏感的荧光报告系结合的小鼠模型越来越多地用于在体内追踪细胞系。在这项研究中，我们使用了两种可诱导的报告菌株，Ai9(iCol2a1) （Ai9xCol2a1-creER(T2)）来追踪封闭骨折模型中骨再生过程中软骨祖细胞的贡献，Ai9(iUBC) （Ai9xUBC-creER(T2)）来研究诱导局部重组的方法。通过与Ai9同窝对照以及未给他莫昔芬的诱导型报告小鼠进行比较，我们发现CreER（T2）系统存在显著的渗漏，特别是在两系的骨髓中。这些研究强调了与高敏感报告基因相关的挑战，这些报告基因可能在表达CreER（T2）融合的组织中不经诱导而被激活。对Ai9（iCol2a1）菌株生长板的检查显示，骨软骨谱系的细胞（细胞与软骨细胞和成骨细胞标记共染色）被tdTom报告基因标记。然而，在Ai9（iCol2a1）小鼠愈合骨折中没有发现这种标记。尝试在Ai9（iUBC）菌株中使用肌肉注射4-羟他莫昔芬标记单个肢体，结果导致整个动物的完整标记，与腹腔注射相当。虽然解释起来很有挑战性，但这些数据仍然提供了关于这些诱导报告者模型的局限性的信息，并证明在未来使用此类模型的研究中谨慎和广泛的控制是合理的。
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.