Welman Arkadiusz, Barraclough Jane, Dive Caroline
Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, United Kingdom.
Nat Protoc. 2006;1(2):803-11. doi: 10.1038/nprot.2006.117.
Tet-on cell lines engineered to stably express doxycycline (Dox)-regulated reverse transcriptional transactivator (rtTA) have many applications in biomedical research and biotechnology. Unfortunately, construction and maintenance of such cells often proves to be costly, labor intensive and ineffective. Moreover, the Tet-on clones generated using standard methodology were often unstable and frequently displayed significantly changed physiological properties compared with their parental cells. Here we describe an optimized protocol for generation of Tet-on cells. The protocol is based on the use of a recently developed pN1p beta actin-rtTA2S-M2-IRES-EGFP vector (where IRES is an internal ribosome entry site) and permits relatively inexpensive construction of many Tet-on clones with essentially 100% efficiency. The method is well suited for 'difficult' cell lines displaying genetic instability and high levels of epigenetic silencing. The constructed Tet-on cells remain stable with time in the absence of any selection agents, are easy to monitor and preserve the characteristics of parental cells. The protocol can be completed in 2 months.
经基因工程改造以稳定表达强力霉素（Dox）调控的逆转录反式激活因子（rtTA）的Tet-on细胞系在生物医学研究和生物技术领域有诸多应用。遗憾的是，构建和维持此类细胞往往成本高昂、 labor intensive且效率低下。此外，使用标准方法生成的Tet-on克隆通常不稳定，与亲代细胞相比，其生理特性经常发生显著变化。在此，我们描述了一种优化的Tet-on细胞生成方案。该方案基于使用一种最近开发的pN1pβ肌动蛋白-rtTA2S-M2-IRES-EGFP载体（其中IRES是一个内部核糖体进入位点），并允许以相对较低的成本构建许多Tet-on克隆，效率基本达到100%。该方法非常适合显示遗传不稳定性和高水平表观遗传沉默的“难处理”细胞系。构建的Tet-on细胞在没有任何选择剂的情况下随时间保持稳定，易于监测并保留亲代细胞的特征。该方案可在2个月内完成。
