Lin Yu-Jei, Lee Tsung-Lin, Ku Chia-Chi
Graduate Institute of Immunology, National Taiwan University College of Medicine.
Graduate Institute of Immunology, National Taiwan University College of Medicine;
J Vis Exp. 2017 Apr 19(122):55206. doi: 10.3791/55206.
Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined "acufection", denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15ΔE7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15ΔE7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.
皮肤免疫反应失调与多种人类皮肤疾病相关。将免疫相关基因直接导入皮肤组织是在人类疾病小鼠模型中研究皮肤炎症免疫调节的一种引人入胜的方法。在此，我们展示了一种经济高效的方案，可将裸DNA递送至小鼠皮肤并实现转基因表达。该方法被称为“针刺转染”，即通过针刺介导的DNA转染。进行针刺转染时，先将小鼠皮肤用磷酸盐缓冲盐水（PBS）中的DNA灌注，然后用一束针灸针轻轻穿刺，以促进DNA吸收并转染到细胞中。质粒DNA大概被皮肤中的角质形成细胞和树突状细胞（DCs）摄取并表达为蛋白质。针刺本身不会造成皮肤损伤或诱导角质形成细胞活化。针刺转染2天后，在转录和翻译水平均检测到皮肤中转染基因的表达，且可持续7天。开发这种针刺转染方法的主要目的是研究一种先前未定义的IL-15异构体。使用该方法，在皮肤中表达了一种外显子7部分缺失的选择性剪接IL-15异构体（IL-15ΔE7），随后用Toll样受体7（TLR7）激动剂咪喹莫特（IMQ）处理以诱导炎症。针刺转染递送至皮肤的IL-15ΔE7可抑制IMQ诱导的皮肤炎症中的角质形成细胞增殖、表皮厚度和中性粒细胞募集。随着对确定皮肤炎症调节机制的兴趣日益增加，本文所述方案为体内DNA递送的基因枪系统或微接种提供了一种经济高效且通用的替代方法。它可能潜在地有助于发现皮肤中新型基因的功能或用于研究皮肤疾病的新疗法。
