Analysis of the immune response to ectopically expressed allergen gene into epidermis

为了分析皮肤表皮的免疫反应，建立异位性皮炎动物模型，我们建立了表皮角质形成细胞特异性表达绿色荧光蛋白（GFP）的基因打靶小鼠。为了避免在胚胎阶段对异位引入的异种抗原的免疫耐受的诱导，我们应用tet-on系统在四环素的控制下通过表皮角质形成细胞表达GFP。本项目采用了以下方法：1）设计在多西环素存在下转导表皮基底细胞特异性角蛋白5启动子下游的rtTA的构建体，并建立基因靶向小鼠，其中将人生长激素的poly A信号引入rtTA下游（2）构建了膜性GFP构建体和插入绝缘子的SV 40 poly A信号构建体（Tet-EGFP Thy 1）的基因打靶小鼠。我们已经通过Southern印迹分析证实了所设计的构建体被成功地引入到小鼠中。我们现在正在研究分析这些小鼠的免疫反应。

To analyze the immunological response in epidermis of the skin, and to establish the animal model system of atopic dermatitis, we conducted to generate gene-targeting mouse which manifests epidermal keratinocyte specific-expression of green fluorescent protein (GFP). To escape the induction of immunological tolerance to ectopically introduced xenoantigen at the embryonic stage, we applied tet-on system to express GFP by epidermal keratinocytes under the control of tetracycline. Following methods were applied in this project: 1) to design the construct which transduces rtTA, downstream of epidermal basal cell specific keratin 5 promoter, in the presence of doxycycline and to establish the gene targeting- mouse in which a poly A signal of human growth hormone was introduced to downstream of rtTA(K5-rtTA2S-M2), (2) to establish the gene targeting mouse in which membranous GFP construct and SV40 poly A signal construct with insertion of insulator (Tet-EGFP Thy1). We already confirmed the designed constructs were successfully introduced into the mouse by southern blot analysis. We are now under investigation to analyze immune response in these mice.