DNA Repair-on-a-Chip: Spatially Encoded Microwell Arrays

项目摘要/摘要 为了抑制癌症、神经退化、衰老，保持基因组的完整性是必不可少的 以及其他疾病。与基因组保存不一致的是DNA损伤，这可能会导致 突变、序列重排和细胞毒性。DNA损伤是不可避免的，就像DNA 破坏性物质存在于我们的环境和细胞中。中和有害的 在DNA损伤的影响下，我们进化出了复杂的DNA修复系统。现在大家都知道 每一条主要的DNA修复途径都能抑制癌症。此外，由于癌症通常是 使用DNA损伤剂治疗，肿瘤的DNA修复能力并不令人惊讶 调节对化疗的敏感性。尽管它很重要，但DNA损伤的测量 和修复远远不是常规的，主要是由于缺乏可靠和快速的DNA损伤 化验。在这里，通过将工程师、生物学家和 为了满足这一需求，我们建议开发一个平台，用于快速半解析 可广泛分布且易于实现的自动化单细胞DNA损伤定量方法 被公共卫生、学术界、工业界和医学界的研究人员应用。 正如第一阶段提交中所定义的，我们创建并测试了一口96井的原型 并对工程设计和生产装置进行了优化，以 产生空间编码20和96井证明了微孔的补充 含有细胞外基质蛋白(EMPS)的彗星凝胶支持UP细胞的生长 到两周，电磁脉冲不会影响彗星的形成。启用角色化 商业使用的化学品的遗传毒性、环境中发现的化学品或新发现的化学品 开发药物，并在不需要识别的情况下量化DNA修复能力 特定的DNA修复技术。这项提议是为了发展“芯片上的DNA修复” 技术，结合使用基于琼脂糖基的微孔阵列，空间编码细胞 识别、自动化数据处理和细胞外基质蛋白，以优化、验证 并将一系列空间编码的微孔阵列商业化。我们将证明我们 显著地改进了制造工艺(Aim1)，开发了一种大井 前者生产96井和384井CometChips(目标2)，并提出实施 用于数据分析的图形用户界面(目标3)。最后，我们将严格验证这一点 通过分析来自NTP的一系列化合物的遗传毒性效应的新技术 文库研究它们对DNA损伤和修复反应的影响，并揭示个体之间和 DNA损伤反应的细胞间类型差异(目标4)。通过整合传统的 方法在生物学和工程学中，描述了在芯片平台上的DNA修复 代表着重大的技术进步，提供了高吞吐量、客观和 有可能成为DNA损伤的新标准的定量测量 分析。

Project Summary / Abstract Preserving genomic integrity is essential in order to suppress cancer, neurodegeneration, aging and other diseases. At odds with genomic preservation is DNA damage, which can drive mutations, sequence rearrangements and cellular toxicity. DNA damage is unavoidable, as DNA damaging agents are present in our environment and in our cells. To counteract the deleterious effects of DNA damage, we have evolved sophisticated DNA repair systems. It is now known that every major DNA repair pathway suppresses cancer. Furthermore, since cancer is often treated using DNA damaging agents, it is not surprising that the DNA repair capacity of tumors modulates sensitivity to chemotherapy. Despite its importance, measurements of DNA damage and repair are far from routine, primarily due to the lack of reliable and rapid DNA damage assays. Here, by bringing together convergent expertise among engineers, biologists and computer programmers, we propose to meet this need by developing a platform for rapid semi- automated single-cell DNA damage quantification that can be broadly distributed and readily applied by researchers in public health, academia, industry and medicine. As defined in the Phase I submission, we created and tested a prototype for a 96-well CometChip platform and have optimized the engineering design and a production apparatus to produce spatially encoded 20 and 96 well demonstrated that supplementation of the Microwell Comet gels with extracellular matrix proteins (EMPs) supports the growth of human cells for up to two weeks and the EMPs do not impact the formation of comets. To enable characterization of the genotoxicity of chemicals used commercially, those found in the environment or newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair technology. This proposal, to develop the 'DNA Repair on a Chip' technology, combines the use of agarose based Microwell arrays, spatially encoded cellular recognition, automated data processing, and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays. We will demonstrate that we have significantly advanced the manufacturing process (Aim1), have developed a macrowell former to produce 96-well and 384-welll CometChips (Aim 2), and propose the implementation of a graphical user interface for data analysis (Aim 3). Finally, we will rigorously validate this new technology by analyzing the genotoxic effects of a range of compounds from the NTP library for their impact on DNA damage and repair responses and to reveal inter-individual and inter-cell type variation in DNA damage responses (Aim 4). Through the integration of traditional methods in biology and engineering, the DNA Repair on a Chip platform described here represents a significant technological advance, providing high-throughput, objective, and quantitative measurements that have the potential to become a new standard in DNA damage analysis.