Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

Abstract

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.

Do DNA Double-Strand Breaks Drive Aging?

“…Of the tissues analyzed, the most drastic age-related decline in HR was observed in pancreas, lung, and thymus, while more modest declines were observed in spleen and kidney (White et al, 2013). Similar results were obtained for intrachromosomal HR, using a non-functional GFP reporter to measure spontaneous HR in mouse tissue (Sukup-Jackson et al, 2014). In these mice it was shown that recombinant cells accumulate with age in the colon (Sukup-Jackson et al, 2014).…”

“…Similar results were obtained for intrachromosomal HR, using a non-functional GFP reporter to measure spontaneous HR in mouse tissue (Sukup-Jackson et al, 2014). In these mice it was shown that recombinant cells accumulate with age in the colon (Sukup-Jackson et al, 2014). Since HR is more dependent on progressing through S and G 2 phases of the cell cycle, due to reliance of an available sister chromatid, observations of reduced repair efficacy may be more indicative of reduced cellular proliferation in aging tissues, for example, due to the increase in senescent cells across tissues (Wang et al, 2009).…”

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving

“…However, DHR and mutagenesis may be triggered by the same underlying cause, such as increased ROS, as discussed further below. To date, no studies have correlated radiation-induced DHR with mutagenesis or carcinogenesis, so these remain important open questions for the future that may be addressed with advanced mutation detection systems, and transgenic mice such as RaDR-GFP mice developed in the Engelward laboratory (63). …”

Robust homology-directed repair within mouse mammary tissue is not specifically affected by Brca2 mutation

“…Prior estimates have suggested low levels of HDR in tissues29, or were inferred from indirect measures, such as toxicity after exposure to DNA damaging agents and DNA damage-induced foci formation3031. The efficient induction of a DNA lesion at a defined genomic location is a powerful approach to understanding DNA repair, and specifically HDR, within the organism.…”