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

DNA 芯片修复：空间编码微孔阵列

• 批准号: 8647690
• 负责人: Bevin P. Engelward
• 金额: $ 105.59万
• 依托单位: TREVIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8647690
• 关键词: Academia; Aging; Area; Automatic Data Processing; Biological Assay; Biological Preservation; Biology; Caliber; Cells; Chemicals; Comet Assay; Computer software; Computers; Cytoskeleton; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA Sequence Rearrangement; DNA repair protein; Data; Data Analyses; Data Quality; Data Storage and Retrieval; Development; Disease; Dose; Drug Industry; Engineering; Ensure; Environment; Environmental Health; Epidemiologist; Exposure to; Extracellular Matrix Proteins; Feedback; Force of Gravity; Gel; Genomics; Glass; Goals; Growth; Hepatocyte; Human; Image; Image Analysis; In Vitro; Individual; Industry; Institutes; Knowledge; Libraries; Mainstreaming; Malignant Neoplasms; Massachusetts; Measurement; Measures; Medicine; Methodology; Methods; Microscopy; Mutagenicity Tests; Mutagens; Mutation; Nerve Degeneration; Output; Performance; Pharmacologic Substance; Phase; Play; Predisposition; Process; Production; Proteins; Public Health; Quality Control; Research; Research Personnel; Role; Sampling; Sepharose; Series; Services; Shipping; Ships; Site; Slide; Supplementation; System; Technology; Testing; Time; Toxic effect; University of Pittsburgh Cancer Institute; Validation; Variant; base; cancer prevention; cell type; chemotherapy; clinical assay development; design; drug development; drug discovery; engineering design; exposed human population; genotoxicity; graphical user interface; in vivo; killings; manufacturing process; meetings; neoplastic cell; new technology; novel; prevent; programs; prototype; public health relevance; response; tumor

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.

项目总结/摘要 保持基因组的完整性对于抑制癌症、神经退行性变和衰老至关重要 和其它疾病。与基因组保存不一致的是DNA损伤， 突变、序列重排和细胞毒性。DNA损伤是不可避免的， 我们的环境和细胞中都存在有害物质。为了抵消有害的 在DNA损伤的影响下，我们进化出了复杂的DNA修复系统。现在已知 每个主要的DNA修复途径都能抑制癌症。此外，由于癌症通常 使用DNA损伤剂治疗，肿瘤的DNA修复能力降低并不奇怪。 调节对化疗的敏感性。尽管DNA损伤的测量很重要， 和修复远非常规，主要是由于缺乏可靠和快速的DNA损伤 测定。在这里，通过汇集工程师、生物学家和 计算机程序员，我们建议，以满足这一需要，通过开发一个平台，快速半， 自动化单细胞DNA损伤定量，可以广泛分布， 公共卫生、学术界、工业界和医学界的研究人员都在使用。 根据第一阶段提交的定义，我们创建并测试了96孔 CometChip平台，并优化了工程设计和生产设备， 产生空间编码的20和96孔证明了微孔的补充 彗星凝胶与细胞外基质蛋白（EMP）支持人类细胞的生长， 电磁脉冲不会影响彗星的形成。要启用表征，请执行以下操作 商业上使用的化学品的遗传毒性，那些在环境中发现的或新的 开发的药物，并量化DNA修复能力，而不需要确定 DNA修复技术。这项建议，开发'DNA修复芯片' 技术，结合使用基于琼脂糖的微孔阵列，空间编码的细胞， 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微孔阵列商业化。我们将证明， 已经显著地改进了制造工艺（Aim 1），已经开发了一种宏孔， 前者生产96孔和384孔CometChips（目标2），并提出实施方案 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NTP中一系列化合物的遗传毒性效应， 库，以了解它们对DNA损伤和修复反应的影响，并揭示个体间和 DNA损伤反应中的细胞间类型变化（目的4）。通过整合传统 生物学和工程学方法，此处描述的芯片上的DNA修复平台 代表了一个重大的技术进步，提供高通量，客观， 定量测量有可能成为DNA损伤的新标准 分析.