Applications of Fluorescence for Detecting Rare Sequence Rearrangements In Vivo

Wiktor-Brown, Dominika M.; Hendricks, Carrie A.; Olipitz, Werner; Rogers, Arlin B.; Engelward, Bevin P.

doi:10.4161/cc.5.23.3527

Cited by 12 publications

(34 citation statements)

References 56 publications

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“…Previous studies of FYDR positive control mice (which express EYFP from the same promoter and locus as the HR reporter) show that there is little or no expression of EYFP in many tissues (presumably due to silencing), which greatly limits the utility of the FYDR model [65]. While we anticipated that targeting the EGFP direct repeat reporter to a site with ubiquitous expression would overcome this barrier to studies of HR, prior studies of expression at the Rosa26 locus had been done using the Rosa26 promoter [68], whereas the CAG promoter drives the RaDR-GFP transgene.…”

Section: Resultsmentioning

confidence: 99%

“…Taken together, studies using the FYDR mice show that fluorescence detection of HR in vivo provides valuable insights into genetic, environmental and physiological factors that modulate HR [58]–[60], [62], [63]. Importantly, however, only a limited number of tissues can be studied in the FYDR mice as a consequence of poor expression in some tissues (presumably due to the random locus integration following pronuclear injection) [58], [65]. We therefore set out to generate a recombination reporter mouse with broad reporter expression.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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“…Clearly, if EYFP is expressed at lower levels, it will be more difficult to detect recombinant cells. To determine if the increased frequency of recombinant cells in pancreatic tissue is due to increased EYFP expression, we analyzed pancreatic and skin tissues from positive control FYDR-Rec mice that carry the full-length EYFP coding sequence in every cell [52]. EYFP expression was, in fact, statistically significantly higher in pancreatic (~52%) versus skin (~30%) tissues (Fig.…”

Section: Comparison Of Spontaneous Recombinant Cell Frequency In Pancmentioning

confidence: 99%

“…Positive control FYDR-Recombined (FYDR-Rec) mice arose spontaneously from an HR event in a FYDR parental gamete, and all cells carry the full-length EYFP coding sequence [52]. FYDR cohorts had equal ratios of males to females.…”

Section: Animalsmentioning

confidence: 99%

“…One method for determining the in vivo frequency of recombinant cells is to analyze disaggregated tissue by flow cytometry [51,56]. Briefly, for both pancreas and skin we compared fluorescence intensities in disaggregated tissue from negative control and positive control (FYDR-Recombined [52]) mice and established a region (R2) that excludes negative control cells (Fig. 1B, note: identical criteria were used to establish an R2 region for pancreas [51]).…”

Section: Comparison Of Spontaneous Recombinant Cell Frequency In Pancmentioning

confidence: 99%

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Tissue-specific differences in the accumulation of sequence rearrangements with age

et al. 2008

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Mitotic homologous recombination (HR) is a critical pathway for the accurate repair of DNA double strand breaks (DSBs) and broken replication forks. While generally error-free, HR can occur between misaligned sequences, resulting in deleterious sequence rearrangements that can contribute to cancer and aging. To learn more about the extent to which HR occurs in different tissues during the aging process, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which an HR event in a transgene yields a fluorescent phenotype. Here, we show tissue-specific differences in the accumulation of recombinant cells with age. Unlike pancreas, which shows a dramatic 23-fold increase in recombinant cell frequency with age, skin shows no increase in vivo. In vitro studies indicate that juvenile and aged primary fibroblasts are similarly able to undergo HR in response to endogenous and exogenous DNA damage. Therefore, the lack of recombinant cell accumulation in the skin is most likely not due to an inability to undergo de novo HR events. We propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of recombinant cells to persist and clonally expand within tissues.

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Recombinant cells in the lung increase with age via de novo recombination events and clonal expansion

Kimoto

Kay

et al. 2017

Environ and Mol Mutagen

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Homologous recombination (HR) is a critical DNA repair pathway, which is usually error-free, but can sometimes lead to cancer-promoting mutations. Despite the importance of HR as a driver of mutations, the spontaneous frequency of such mutations has proven difficult to study. In order to gain insight to location, cell type, and subsequent proliferation of mutated cells, we utilized the Rosa26 Direct Repeat (RaDR) mice for in situ detection and quantification of recombinant cells in the lung. We developed a method for automated enumeration of recombinant cells in lung tissue using the Metafer 4 slide-scanning platform. The mean spontaneous HR frequencies of the lung tissue in young and aged mice were 2×10 −6 and 30×10 −6 respectively, which is consistent with our previous reports that mutated cells accumulate with age. In addition, by using the capability of Metafer 4 to mark the position of fluorescent cells, we found that recombinant cells from the aged mice formed clusters in the lung tissue, likely due to clonal expansion of a single mutant cell. The recombinant cells primarily consisted of alveolar epithelial type II or club (previously known as Clara) cells, both of which have the potential to give rise to cancer. This approach to tissue image analysis reveals the location and cell types that have undergone HR. Being able to quantify mutant cells in situ within lung tissue opens doors to studies of exposure-induced mutations and clonal expansion, giving rise to new opportunities for understanding how genetic and environmental factors cause tumorigenic mutations.

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Applications of Fluorescence for Detecting Rare Sequence Rearrangements In Vivo

Cited by 12 publications

References 56 publications

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

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

Tissue-specific differences in the accumulation of sequence rearrangements with age

Recombinant cells in the lung increase with age via de novo recombination events and clonal expansion

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