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.

中文翻译：

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空间受限的长期转基因表达在发育中的皮肤中，用于研究表皮发育和感觉神经的相互作用。

皮肤发育在时间上与其感觉神经紧密相关，后者由背根神经节（DRG）轴突的外围分支组成。各种研究表明，皮肤为感觉轴突产生了远距离引诱剂。但是，指导提示的确切身份仍不清楚。为了揭示控制DRG轴突引导和靶向的详细分子机制，需要在特定时间点对特定皮肤层进行操作。为了测试可以在特定时间点在特定皮肤层中表达的多种引诱剂，我们将子宫电穿孔与Tol2转座子系统结合使用，以诱导发育中的小鼠皮肤中长期转基因表达，包括高度增殖的表皮干细胞（基底层）及其后代（棘突和颗粒层细胞）。将质粒溶液注射到尽可能靠近后爪的足底表面。立即将电脉冲穿过胚胎，将质粒DNA转导至后爪皮肤细胞。平衡结果测量包括：胚胎存活，转染效率和转基因整合入宿主细胞的效率，我们发现IUE在E13.5上执行最佳，并使用34V电穿孔电压。在对角质形成细胞和感觉轴突标记物的胚胎和产后早期皮肤组织切片进行免疫染色后，我们观察到轴突生长到皮肤表皮层中，包括表达EGFP的区域。因此，