A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture
A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture
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DOI：
10.1016/s0378-1119(02)00979-4
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发表时间：
2002-10-02
期刊：
影响因子：
3.5
通讯作者：
Stewart, AF
中科院分区：
文献类型：
--
作者：
Anastassiadis, K;Kim, J;Stewart, AF
已经描述了在哺乳动物细胞中调节稳定转基因表达的几种策略。这些策略有不同的优点和缺点，但它们都有一个共同的问题，即应用的可预测性。在这里，我们使用配体诱导转基因表达的领先策略——四环素阻遏系统来解决这个问题。最初，我们发现 48 个检查中最稳定的克隆仅表现出 6 倍的诱导能力。因此，我们寻找可以提高成功机会的补充和修改。我们记录了三个重要方面；首先，使用哺乳动物密码子优化的四环素阻遏基因；其次，将类固醇激素受体配体结合结构域添加到四环素阻遏物-病毒体蛋白16融合蛋白激活剂中；第三，在tet操纵子/转基因盒的侧翼添加来自鸡β-珠蛋白基因座的绝缘体元件。通过纳入这三个设计特征，18/18 克隆显示出低基础和高诱导型 (>50 X) 表达。 (C) 2002 Elsevier Science B.V. 保留所有权利。
Several strategies for regulated stable transgene expression in mammalian cells have been described. These strategies have different strengths and weaknesses, however they all share a common problem, namely predictability in application. Here we address this problem using the leading strategy for ligand inducible transgene expression, the tetracycline repressor system. Initially, we found the best stable clone out of 48 examined showed only 6-fold inducibility. Hence we looked for additions and modifications that improve the chances of a successful outcome. We document three important aspects; first, use of a mammalian codon-optimized tetracycline repressor gene; second, addition of a steroid hormone receptor ligand binding domain to the tetracycline repressor-virion protein 16 fusion protein activator; third, flanking the tet-operator/transgene cassette with insulator elements from the chicken beta-globin locus. By inclusion of these three design features, 18/18 clones showed low basal and highly inducible (>50 X) expression. (C) 2002 Elsevier Science B.V. All rights reserved.