title: 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.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Sukup-Jackson
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Orsolya
authors.initials: O
authors.lastname: Kiraly
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Kay
authors.affiliation: Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.
authors.forename: Li
authors.initials: L
authors.lastname: Na
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Rowland
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Winther
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Danielle N
authors.initials: DN
authors.lastname: Chow
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kimoto
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Matsuguchi
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Vidya S
authors.initials: VS
authors.lastname: Jonnalagadda
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Vilena I
authors.initials: VI
authors.lastname: Maklakova
authors.affiliation: Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.
authors.forename: Vijay R
authors.initials: VR
authors.lastname: Singh
authors.affiliation: Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.
authors.forename: Dushan N
authors.initials: DN
authors.lastname: Wadduwage
authors.affiliation: Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.
authors.forename: Jagath
authors.initials: J
authors.lastname: Rajapakse
authors.affiliation: 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.
authors.forename: Peter T C
authors.initials: PT
authors.lastname: So
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Lara S
authors.initials: LS
authors.lastname: Collier
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America; Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.
authors.forename: Bevin P
authors.initials: BP
authors.lastname: Engelward
mesh_terms: D000367:Age Factors; D000375:Aging; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001921:Brain / Q000166:cytology; D003106:Colon / Q000166:cytology; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics*; D042822:Genomic Instability / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics*; D059765:Homologous Recombination / Q000235:genetics*; D008099:Liver / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010179:Pancreas / Q000166:cytology; D022661:RNA, Untranslated / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D008164:Luminescent Proteins; D022661:RNA, Untranslated; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins