Spatially restricted long‐term transgene expression in the developing skin used for studying the interaction of epidermal development and sensory innervation
Spatially restricted long‐term transgene expression in the developing skin used for studying the interaction of epidermal development and sensory innervation
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DOI：
10.1111/dgd.12603
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发表时间：
2019-04
期刊：
影响因子：
4.6
通讯作者：
Carmen C. M. Chan;H. Kamiguchi;T. Shimogori
中科院分区：
文献类型：
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作者：
Carmen C. M. Chan;H. Kamiguchi;T. Shimogori
皮肤的发育与其感觉神经支配在时间上紧密协调，感觉神经支配由背根神经节（DRG）轴突的外周分支组成。各种研究表明，皮肤对感觉轴突产生长距离的引诱剂。然而，指导线索的确切身份仍然不清楚。为了揭示控制DRG轴突引导和靶向的详细分子机制，需要在特定时间点操纵特定皮肤层。为了测试可以在特定时间点在特定皮肤层中表达的各种引诱剂，我们将子宫内电穿孔与Tol 2转座子系统相结合，以诱导发育中的小鼠皮肤中的长期转基因表达，包括高度增殖的表皮干细胞（基底层）及其后代（棘层和颗粒层细胞）。将质粒溶液尽可能靠近后爪足底表面注射。立即，电脉冲通过胚胎将质粒DNA导入后爪皮肤细胞。平衡结果测量，包括：胚胎存活率，转染效率，和转基因整合到宿主细胞的效率，我们发现，IUE是最好的E13.5上进行，并使用34 V的电穿孔电压。在对胚胎和出生后早期皮肤组织切片的角质形成细胞和感觉轴突标记物进行免疫染色后，我们观察轴突生长到皮肤表皮层，包括表达EGFP的区域。因此，这种方法是有用的研究轴突生长和表皮细胞分裂/分化之间的相互作用。
Skin development is tightly temporally coordinated with its sensory innervation, which consists of the peripheral branches of the dorsal root ganglion (DRG) axons. Various studies suggest that the skin produces a long‐range attractant for the sensory axons. However, the exact identity of the guidance cue(s) remains unclear. To reveal the detailed molecular mechanism that controls DRG axon guidance and targeting, manipulation of specific skin layers at specific time points are required. To test a variety of attractants that can be expressed in specific skin layers at specific timepoints, we combined in utero electroporation with the Tol2 transposon system to induce long‐term transgene expression in the developing mouse skin, including in the highly proliferative epidermal stem cells (basal layer) and their descendants (spinous and granular layer cells). The plasmid solution was injected as close to the hindpaw plantar surface as possible. Immediately, electric pulses were passed through the embryo to transduce the plasmid DNA into hindpaw skin cells. Balancing outcome measurements including: embryo survival, transfection efficiency, and the efficiency of transgene integration into host cells, we found that IUE was best performed on E13.5, and using an electroporation voltage of 34V. After immunostaining embryonic and early postnatal skin tissue sections for keratinocyte and sensory axon markers, we observe the growth of axons into skin epidermal layers including areas expressing EGFP. Therefore, this method is useful for studying the interaction between axon growth and epidermal cell division/differentiation.