Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter
Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter
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DOI：
10.1046/j.1523-1747.2000.00144.x
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发表时间：
2000-11-01
影响因子：
6.5
通讯作者：
Glick, A
中科院分区：
文献类型：
--
作者：
Diamond, I;Owolabi, T;Glick, A
为了在小鼠表皮中产生基因的条件表达，我们已经产生了转基因小鼠系，其中四环素调节的转录反式激活因子tTA和rTA与牛角蛋白5启动子连接。将反式激活因子系与tetOlacZ指示剂系杂交以测试体内反式激活。在不存在多西环素的情况下，K5/tTA系诱导表皮中的β-半乳糖苷酶活性，其水平比对照高500倍，并且口服和局部多西环素引起β-半乳糖苷酶mRNA水平和酶活性的剂量和时间依赖性抑制。在K5/rTA细胞系中，多西环素诱导的β-半乳糖苷酶活性高3- 50倍，这取决于创始细胞系，并且这发生在给药后24-48小时内。所有细胞系的组织化学分析均将β-半乳糖苷酶表达定位于表皮的基底层和毛囊的外根鞘以及其他角蛋白5阳性组织。在几个K5/rTA系中，皮肤特异性反式激活仅限于毛囊。用12-O-十四烷酰基-佛波醇-13-乙酸酯处理这些双转基因小鼠，引起β-半乳糖苷酶标记的细胞从毛囊通过毛囊间表皮快速迁移，证明了这种特异性双转基因对表皮中命运作图细胞的有用性。这些结果表明，四环素调控系统在小鼠表皮中产生有效的条件性基因表达，并表明它应该能够在发育和与表皮相关的特定病理条件下抑制和激活外源基因。
To produce conditional expression of genes in the mouse epidermis we have generated transgenic mouse lines in which the tetracycline-regulated transcriptional transactivators, tTA and rTA, are linked to the bovine keratin 5 promoter. The transactivator lines were crossed with the tetOlacZ indicator line to test for transactivation in vivo. In the absence of doxycycline, the K5/tTA line induced beta -galactosidase enzyme activity in the epidermis at a level 500-fold higher than controls, and oral and topical doxycycline caused a dose- and time-dependent suppression of beta -galactosidase mRNA levels and enzyme activity. In the K5/rTA lines, doxycycline induced beta -galactosidase activity between 3- and 50-fold higher depending on the founder line, and this occurred within 24-48 h after dosing. Histochemical analysis of all lines localized beta -galactosidase expression to the basal layer of the epidermis and the outer root sheath of the hair follicle, as well as other keratin 5 positive tissues. In several K5/rTA lines, skin-specific transactivation was restricted to the hair follicle. Treatment of these double transgenic mice with 12-O-tetradecanoyl-phorbol-13-acetate caused rapid migration of beta -galactosidase marked cells from the hair follicle through the interfollicular epidermis, demonstrating the usefulness of this specific double transgenic for fate mapping cells in the epidermis. These results show that the tetracycline regulatory system produces effective conditional gene expression in the mouse epidermis, and suggest that it should be amenable to suppression and activation of foreign genes during development and specific pathologic conditions relevant to the epidermis.