Developing novel methods to measure DNA repair capacity in human populations

描述 摘要 该项目旨在开发一种新的、多路高通量的方法来测量来自不同个体的细胞修复各种DNA损伤的能力，代表我们环境中存在的试剂造成的损伤，以及化疗药物引起的损伤。测量这些能力将整合DNA修复基因中的基因型的影响，以及这些基因表达水平的差异的影响。我们将开发一个表达载体文库，每个表达载体包含一种不同荧光蛋白的基因，每个基因都经过改造，包含一种特定类型的DNA损伤。我们将把这些质粒的混合物导入细胞，随着时间的推移，将监测每种DNA损伤的修复情况，因为随着DNA损伤(已知抑制转录)的修复，荧光蛋白的表达将增加。可以重复测量单个细胞的荧光成像方法将被开发出来。最终目标是开发该方法，使其成为常规应用于人类血液样本的标准临床测试，并将用于监测许多不同人类细胞类型的DNA修复能力(例如，正在接受化疗的癌症患者的骨髓细胞，以及正在接受治疗的癌细胞)。从长远来看，使用干细胞分化来帮助确定每个个体的一组组织的DNA修复能力，可以提供关于该个体对环境暴露的敏感性，或个体抵御放射和化疗毒副作用的能力的重要信息。 公共卫生相关性 在我们的一生中，我们无情地暴露在对我们的遗传物质造成损害的化学和物理制剂中。许多这样的物质存在于我们的自然和人造环境中，有些是由细胞内源性产生的，还有

DESCRIPTION Abstract This project seeks to develop a novel, multiplexed high-throughput method to measure the capacity of cells from different individuals to repair a wide variety of DNA damages, representative of damage induced by agents present in our environment, and representative of damage induced by chemotherapeutic agents. Measuring these capacities will integrate the influence of genotype across DNA repair genes and the influence of variations in the expression level for these genes. We will develop a library of expression vectors each containing a gene for a different fluorescent protein, with each gene having been engineered to contain a specific kind of DNA lesion. We will transfect a mixture of these plasmids into cells and the repair of each kind of DNA lesion will be monitored over time because it will be reported by increased expression of fluorescent protein as the DNA lesions (known to inhibit transcription) are repaired. Fluorescent imaging methods allowing repeated measurements of individual cells will be developed. The ultimate goal is to develop the methodology such that it will become a standard clinical test applied to human blood samples as a matter of routine and that it will be used to monitor DNA repair capacity in many different human cell types (e.g., bone marrow cells of cancer patients undergoing chemotherapy, as well as the cancer cells being treated). In the long term using stem cell differentiation to aid in determining DNA repair capacity for a panel of tissues from each individual could provide important information on the susceptibility of that individual to environmental exposures, or the ability of an individual to withstand the toxic side effects of radiation and chemotherapy. Public Health Relevance Throughout our lifetime we are relentlessly exposed to chemical and physical agents that cause damage to our genetic material. Many such agents are present in our natural and manmade environment, some are produced endogenously by cells, and othe