Sukup-Jackson Michelle R, Kiraly Orsolya, Kay Jennifer E, Na Li, Rowland Elizabeth A, Winther Kelly E, Chow Danielle N, Kimoto Takafumi, Matsuguchi Tetsuya, Jonnalagadda Vidya S, Maklakova Vilena I, Singh Vijay R, Wadduwage Dushan N, Rajapakse Jagath, So Peter T C, Collier Lara S, Engelward Bevin P
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.
PLoS Genet. 2014 Jun 5;10(6):e1004299. doi: 10.1371/journal.pgen.1004299. eCollection 2014 Jun.
Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.
同源重组（HR）对于双链断裂和断裂的复制叉的修复至关重要。尽管HR大多无差错，但抑制或诱导HR的内在或环境条件会导致基因组不稳定。尽管其在致癌过程中很重要，但由于我们在体内检测HR的能力有限，对哺乳动物组织中的HR了解甚少。在此，我们描述了一种小鼠模型，其中一个直接重复HR底物靶向普遍表达的Rosa26位点。在Rosa26直接重复-绿色荧光蛋白（RaDR-GFP）小鼠中，两个截短的EGFP表达盒之间的HR可产生荧光信号。内部图像分析软件提供了一种在完整组织内定量重组事件的快速方法，并且可以通过流式细胞术评估重组细胞的频率。对11个组织的比较表明，各组织中重组细胞的频率相差两个以上数量级，其中大脑中的HR最低。此外，结肠中从头重组事件随年龄积累，表明该小鼠模型可用于研究慢性暴露对基因组稳定性的影响。暴露于N-甲基-N-亚硝基脲（一种类似于癌症化疗药物替莫唑胺的烷化剂）表明，结肠、肝脏和胰腺易受DNA损伤诱导的HR影响。最后，对潜在细胞类型的组织学分析表明，胰腺腺泡细胞和肝脏肝细胞会发生HR，并且在结肠体细胞干细胞中也能特异性检测到HR。综上所述，RaDR-GFP小鼠模型为组织和年龄如何影响对HR的易感性提供了新的认识，并有助于未来对调节哺乳动物HR的遗传、环境和生理因素的研究。
