A dual-activation, adenoviral-based system for the controlled induction of DNA double-strand breaks by the restriction endonuclease SacI

作者：Maslov Alexander Y^*; Metrikin Maya; Vijg Jan

来源：Biotechniques, 2009, 47(4): 847-853.

DOI：10.2144/000113237

• DNA damage
• restriction endonuclease
• double-strand breaks
• aging

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.

• 出版日期2009-10