Homologous recombination (HR) is a DNA repair process critical for maintaining genomic integrity. HR is generally beneficial, but over‐ or under‐utilization of HR can lead to can lead to deleterious rearrangements and cancer. To study HR, our laboratory previously developed the Fluorescent Yellow Direct Repeat (FYDR) mouse in which HR yields a fluorescent signal.We have utilized this mouse to study the interplay between HR and non‐homologous end‐joining (NHEJ) by knocking out the Ku86 protein. Both pathways are known to repair double strand breaks (DSB); preventing NHEJ in Ku86−/− mice resulted in an increase in HR. The newly proposed alternative end‐joining pathway was explored by knocking down the repair protein Ercc1. The Ercc1−/Δ mice showed an increase in HR in the pancreas, confirming in vivo an alternative DSB repair pathway and implicating ERCC1 in this pathway.While an excellent tool, the FYDR mouse is limited to study of HR in the pancreas and skin. We created the RADR (Recombination at a Direct Repeat) mouse, an improved model that has enabled our study of HR in a wide range of tissues including gut and liver. We can now study DNA damage and HR in multiple tissues in response to a treatment or exposure.Grant Funding Source : NIEHS Grant T32‐ES07020, NSF GRF, NIH Grant# P01‐CA026731‐31, R33‐CA112151‐01A2
Homologous recombination (HR) is a DNA repair process that is critical for maintaining genomic integrity. HR is necessary for survival of vertebrate cells, but over‐ or under‐utilization of HR can lead to deleterious rearrangements and cancer. To study HR in vivo, we created the RADR (Rosa26 Direct Repeat) mouse that has enabled study of HR in a variety of tissues. The RADR mouse harbors two truncated EGFP genes integrated in the Rosa26 locus. Repair via HR at the substrate can yield a full‐length EGFP gene, resulting in a fluorescent cell. The frequency of HR can be estimated by flow cytometry or visualized in situ. The RADR mouse enables studies of DNA damage and repair in response to endogenous and exogenous factors in multiple tissues, which has never before been possible. We observed the accumulation of recombinant cells in the colon, liver and pancreas with age; this correlates with our understanding of age as a risk factor for cancer. We have also crossed the RADR mice with GPT‐Δ mice, enabling quantification of point mutations and small deletions. We crossed these mice with Rag2−/− mice allowing the study of sequence changes with enhanced innate immune response. In ongoing studies, we are investigating the impact of environmentally induced inflammation on susceptibility to large‐scale sequence rearrangements, point mutations and small deletions in multiple tissues. This work is supported by the NCI, NIEHS, NSF GRF.Grant Funding Source: NSF GRF, NCI, NIEHS
The ability of cells to repair damaged DNA via homologous recombination (HR) is crucial to maintaining the fidelity of genomic information. While generally beneficial, HR can cause tumorgenic sequence rearrangements.Our laboratory has developed a method for measuring HR frequency in the mouse. The Fluorescent Yellow Direct Repeat (FYDR) mice harbor incomplete repeat copies of the EYFP coding sequence. In the event of HR repair at the substrate, a complete EYFP sequence can result, leading to a fluorescent cell visible via flow cytometry.In the FYDR mice, the EYFP substrate is not adequately expressed in all tissue types, preventing study of HR. To study previously inaccessible tissues, we have developed a new vector targeted at the ubiquitously expressed Rosa26 locus. We describe a new targeting vector consisting of tandem EGFP cassettes lacking essential sequences and initial characterization of the model.We will use this model for many studies, specifically genomic stability in tissues susceptible to inflammation‐induced cancer. Knowledge of the relationship between exposures and HR will help us identify key factors modulating cancer susceptibility. NSF GRF, NIH# P01‐CA26731, R33‐CA112151‐01A2Grant Funding Source: R33‐Ca112151‐01A2; NSF GRF, NIH Grant# P01‐CA26731
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