Abstract
Ionizing radiation induces 8-Oxoguanine (8-Oxo-Gua) which is a typical oxidative DNA adduct that is involved in gene mutations and aging. We have developed a new experimental system as a model of low-dose ionizing radiation exposure, which can be used to determine the mutagenicity of synthetic DNA adducts in the genome of human lymphoblastoid TK6 cells. We first prepared a targeting vector that contained a single 8-Oxo-Gua lesion and introduced it into the genome of a TSCER122 cell line (constructed from TK6 cells), after which it was incubated in selective media for 2 weeks. Next, genomic DNAs were obtained from 8-Oxo-Gua-integrated clones and sequenced around the 8-Oxo-Gua site. As a result, when an unmodified Gua vector (Control) was transfected, the Gua site of all of the 22 clones paired with Cyt, the correct base. Following 8-Oxo-Gua vector transfection, the 8-Oxo-Gua lesions of 5 (15%) or 1 (3%) of the 33 clones formed mispairing with Ade or Thy, respectively. In summary, we developed a new experimental system for determining the mutagenicity of DNA adducts introduced by targeting and found that a single 8-Oxo-Gua lesion induced point mutations in the human genome.
