The impact of age, exposure and genetics on homologous recombination at the engineered repeat sequence in mice

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references.Mitotic homologous recombination is a critical pathway for the repair of DNA double-strand breaks and broken replication forks. Although homologous recombination is generally error-free, recombination between misaligned sequences can lead to deleterious sequence rearrangements, and conditions that stimulate homologous recombination are associated with an increased risk of cancer. To study homologous recombination in vivo, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which a homologous recombination event at a transgene yields a fluorescent cell. To study homologous recombination using FYDR mice, we developed one- and two-photon in situ imaging techniques that reveal both the frequency and the sizes of isolated recombinant cell clusters within intact pancreatic tissue. We then applied these tools to analyze the effects of cancer risk factors such as exposure, genetic predisposition and age on homologous recombination in vivo. To determine the effect of exposure to exogenous carcinogens on homologous recombination, FYDR mice were treated with two different chemotherapeutic agents, cisplatin and mitomycin-C.(cont.) Results show that exposure to these DNA damaging agents causes an induction of recombinant pancreatic cells in vivo, indicating that homologous recombination is an active repair pathway in adult pancreatic cells and that exposure to certain carcinogens stimulates recombinational repair. As a first step towards exploring the effect of genetic predisposition to genomic instability on homologous recombination in vivo, FYDR mice were crossed with mice carrying a defect in p53, a critical tumor suppressor that is mutated in almost 50% of all human tumors. Although loss of p53 is known to promote genomic instability, results show that p53 status does not significantly affect the spontaneous recombinant cell frequency in the pancreas in vivo or the rate of homologous recombination in cultured fibroblasts in vitro. Age is a risk factor for many types of cancers. Here we examined the effect of age on homologous recombination in two tissues of FYDR mice, pancreas and skin. In the pancreas, a dramatic accumulation of recombinant cells is seen with age, resulting from both de novo recombination events and clonal expansion of recombinant cells. In contrast, the skin shows no increase in recombinant cell frequency with age.(cont.) In vitro studies using primary fibroblasts indicate that the ability to undergo homologous recombination in response to endogenous and exogenous DNA damage does not significantly change with age, suggesting that these skin cells are able to undergo de novo homologous recombination events in aged mice. Thus, we propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of these cells to persist and clonally expand within the tissue. To further characterize the FYDR mice as a tool for studying homologous recombination, we exploited positive control FYDR-Recombined mice in which all cells carry the full-length coding sequence for enhanced yellow fluorescent protein. Studies show that expression of the FYDR transgene varies among mice, among tissues, and even among cells within a tissue. However, the variation in FYDR expression does not significantly change with age or exposure to exogenous carcinogens. Furthermore, positive control mice reveal that several tissues, in addition to the pancreas and skin, may be amenable for studying homologous recombination in the FYDR mice.(cont.) Thus, our studies demonstrate that FYDR mice combined with in situ imaging technology provide powerful tools to study the effects of cancer risk factors on homologous recombination in vivo. Ultimately, by applying these techniques to study additional cancer risk factors, we may better understand the relationship between DNA damage, homologous recombination and cancer.by Dominika M. Wiktor-Brown.Ph.D