Greehey Children's Cancer Research Institute, The University of Texas Health Science Center at San Antonio, 8403 Floyd Curl Drive, San Antonio, TX 78229, USA; Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.
DNA Repair (Amst). 2013 Nov;12(11):912-21. doi: 10.1016/j.dnarep.2013.08.005. Epub 2013 Sep 10.
Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures.
我们对同源重组的大部分理解，以及这些过程的工作模型的发展，都来自于对酵母和果蝇等模式生物以及哺乳动物系统的广泛研究，通过研究体外诱导的双链断裂的修复或暴露于遗传毒性试剂后的修复。因此，我们着手将这项体外工作扩展到体内同源重组小鼠模型中，以研究不同作用模式的 DNA 损伤剂是否可以诱导体细胞变化。我们使怀孕的母鼠暴露于 DNA 损伤剂中，在胚胎发育的特定时间造成各种损伤。为了监测同源重组的频率，我们使用了成熟的视网膜色素上皮细胞（RPE）pink-eyed 不稳定（pink-eyed unstable，PE）测定法。同源重组导致编码 Oca2 基因的 70kb 重复片段缺失，从而使该基因具有功能，并可在视网膜色素上皮中观察到色素斑点。我们观察到，与喜树碱和羟基脲暴露后可检测到的反转事件频率降低形成对比的是，顺铂、甲磺酸甲酯、乙磺酸乙酯、3-氨基苯甲酰胺、博来霉素和依托泊苷处理后所得的后代中色素斑点的出现频率增加。来自如此广泛的不同作用的损伤剂的体细胞基因组重排意味着即使是短期的宫内暴露也可能会产生长期的潜在影响。
