Co-localization of Cell Lineage Markers and the Tomato Signal
Co-localization of Cell Lineage Markers and the Tomato Signal
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DOI：
10.3791/54982
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发表时间：
2016-12-01
影响因子：
1.2
通讯作者：
Feng, Jian Q.
中科院分区：
文献类型：
--
作者：
Jing, Yan;Hinton, Robert J.;Feng, Jian Q.
细胞谱系追踪系统主要用于发育生物学研究。使用Cre重组酶可以激活特定细胞系和所有后代中的报告基因。在这里，我们使用细胞谱系追踪技术，利用两种Cre， Col10a1-Cre和agrecan -Cre(ERT2) (Agg-Cre(ERT2))，与Rosa26（tdTomato）杂交，证明软骨细胞在长骨和下颌髁发育过程中直接转化为成骨细胞和骨细胞。Col10和聚集蛋白都是公认的软骨细胞标志物。在此基础上，我们开发了一种新的方法-细胞谱系追踪结合荧光免疫组织化学-通过分析特定细胞标志物的表达来确定细胞命运。使用Runx2（早期成骨细胞标志物）和牙本质基质蛋白1 （DMP1，晚期成骨细胞标志物）鉴定软骨细胞来源的骨细胞及其分化状态。这种结合不仅拓宽了细胞谱系追踪的应用，而且简化了复合小鼠的生成。更重要的是，同时显示亲本细胞子代的数量、位置和分化状态，提供比单独的细胞谱系追踪更多的信息。综上所述，细胞谱系示踪技术和免疫荧光技术的联合应用是研究体内细胞生物学的有力工具。
The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-Cre(ERT2) (Agg-Cre(ERT2)), crossed with Rosa26(tdTomato). Both Col10 and aggrecan are well-recognized markers for chondrocytes.On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.