Generation of a mammalian cell line stably expressing a tetracycline-regulated epitope-tagged human androgen receptor: implications for steroid hormone receptor research.
Generation of a mammalian cell line stably expressing a tetracycline-regulated epitope-tagged human androgen receptor: implications for steroid hormone receptor research.
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稳定表达四环素调节表位标记的人类雄激素受体的哺乳动物细胞系的产生：对类固醇激素受体研究的影响。
DOI：
10.1006/abio.2000.4960
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发表时间：
2001
影响因子：
2.9
通讯作者：
Fondell,JD
中科院分区：
文献类型：
--
作者：
Wang,Q;Fondell,JD
雄激素受体（AR）是一种雄激素激活的转录因子，调节参与分化、发育和维持男性生殖功能的基因表达。为了建立一个有用的模型系统，用于研究AR作用的分子机制，我们产生了一个HeLa衍生的细胞系（称为E19），稳定表达人AR。由于培养细胞中AR的过度表达可能具有细胞毒性，我们将AR表达置于四环素调控启动子的控制下。稳定表达的AR还含有N-末端FLAG-表位标签（f：AR），其提供了用于免疫纯化的有利方法。我们发现，在E19细胞中的f：AR表达可以通过改变生长培养基中四环素或其化学衍生物强力霉素的浓度来精确调节。E19表达的f：AR的功能活性在体内通过其以雄激素依赖性方式激活瞬时转染的AR报告基因的能力来证明，并且在体外通过其使用DNA迁移率变化测定特异性结合AR反应元件的能力来证明。我们进一步表明，在雄激素刺激的E19细胞的f：AR显着磷酸化和coimmunopurifies与转录辅激活CREB结合蛋白（CBP）。这些研究结果对类固醇受体研究和受体共调节因子的识别的影响将进行讨论。
The androgen receptor (AR) is hormone-activated transcription factor that regulates the expression of genes involved in differentiation, development, and maintenance of male reproductive functions. To establish a useful model system for studying molecular mechanisms of AR action, we generated a HeLa-derived cell line (termed E19) that stably expresses human AR. Because overexpression of AR in cultured cells can be cytotoxic, we placed AR expression under the control of a tetracycline-regulated promoter. The stably expressed AR also contains an N-terminal FLAG-epitope tag (f:AR) that provides an advantageous method for immunopurification. We show that f:AR expression in E19 cells can be precisely modulated by varying the concentration of tetracycline or its chemical derivative doxycycline in the growth media. The functional activity of E19-expressed f:AR is demonstrated in vivo by its ability to activate transiently transfected AR reporter genes in an androgen-dependent manner, and in vitro by its ability to specifically bind AR-response elements using DNA-mobility shift assays. We further show that f:AR in androgen-stimulated E19 cells is markedly phosphorylated and coimmunopurifies with the transcriptional coactivator CREB-binding protein (CBP). The implications of these findings on steroid receptor research and the identification of receptor coregulatory factors will be discussed.