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PLoS Genet. 2014 Jun 5;10(6):e1004299. doi: 10.1371/journal.pgen.1004299. eCollection 2014 Jun.

Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

Author information

1
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
2
Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.
3
School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
4
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America; Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore; Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
5
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America; Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.

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.

PMID:
24901438
PMCID:
PMC4046920
DOI:
10.1371/journal.pgen.1004299
[Indexed for MEDLINE]
Free PMC Article

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