Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Biotechniques. 2009 Oct;47(4):847-54. doi: 10.2144/000113237.
Spontaneous damage to DNA is frequent and may lead to cell death, cell senescence, or mutations. DNA double-strand breaks (DSBs) are of special interest because they are highly toxic and have been implicated in neurodegeneration, cancer, and aging. Until now, there has not been a reliable system allowing tunable induction of random DSBs without affecting other macromolecules or cell functions. Here, we describe an adenoviral-based, doxycycline-mediated, and tamoxifen-dependent system for quantitative introduction of DSBs in mammalian cells. We generated a single adenoviral vector containing a tet-inducible, composite SacI restriction endonuclease/estrogen receptor (ERT2) gene, and a constitutively expressed reverse transactivator (rtTA) gene. Transduced mouse embryonic fibroblasts-as well as mouse liver cells in vivo-demonstrated a high level of DSBs in response to treatment with doxycycline and tamoxifen. We show that the amount of induced DSBs can be titrated by doxycycline dose and duration of treatment. This system should be useful for studying the processing of randomly induced DSBs and their effects on cell fate, without the side effects normally associated with radiation or chemical treatment.
DNA 的自发性损伤很频繁，可能导致细胞死亡、细胞衰老或突变。DNA 双链断裂（DSBs）特别引人注目，因为它们具有高度的毒性，并与神经退行性疾病、癌症和衰老有关。到目前为止，还没有一种可靠的系统能够在不影响其他大分子或细胞功能的情况下，对随机 DSBs 进行可调节的诱导。在这里，我们描述了一种基于腺病毒的、强力霉素介导的、他莫昔芬依赖性的哺乳动物细胞 DSBs 定量引入系统。我们生成了一个包含 tet 诱导的复合 SacI 限制性内切酶/雌激素受体（ERT2）基因和一个组成型表达的逆转录激活剂（rtTA）基因的单腺病毒载体。转导的小鼠胚胎成纤维细胞以及体内的小鼠肝细胞对强力霉素和他莫昔芬的处理表现出高水平的 DSBs。我们表明，诱导的 DSBs 数量可以通过强力霉素剂量和处理时间来滴定。该系统对于研究随机诱导的 DSBs 的处理及其对细胞命运的影响应该是有用的，而不会产生通常与辐射或化学处理相关的副作用。
