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: 8743205
• 负责人: Bevin P. Engelward
• 金额: $ 44.41万
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8743205
• 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

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：为了抑制癌症、神经变性、衰老和其他疾病，保持基因组完整性至关重要。 DNA 损伤与基因组保存相矛盾，它会导致突变、序列重排和细胞毒性。 DNA 损伤是不可避免的，因为 DNA 损伤剂存在于我们的环境和细胞中。为了抵消 DNA 损伤的有害影响，我们进化出了复杂的 DNA 修复系统。现在已知每条主要的 DNA 修复途径都可以抑制癌症。此外，由于癌症通常使用 DNA 损伤剂进行治疗，因此肿瘤的 DNA 修复能力调节对化疗的敏感性也就不足为奇了。尽管 DNA 损伤和修复的测量很重要，但它远非常规，这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之间的专业知识，我们建议通过开发一个快速半自动化单细胞 DNA 损伤定量平台来满足这一需求，该平台可以广泛分发并易于公共卫生研究人员应用， 学术界、工业界和医学界。 正如第一阶段提交中所定义的，我们创建并测试了 96 孔 CometChip 平台的原型，并优化了工程设计和生产装置，以生产空间编码的 20 和 96 孔，证明了用细胞外基质补充 Microwell Comet 凝胶蛋白质 (EMP) 支持人体细胞生长长达两周，并且 EMP 不会影响彗星的形成。能够表征商业使用的化学品、环境中发现的化学品或新开发的药物的遗传毒性，并量化 DNA 修复能力，而无需识别特定的 DNA 修复技术。该提案旨在开发“芯片上的 DNA 修复”技术，结合使用基于琼脂糖的微孔阵列、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、验证和商业化一系列空间编码微孔阵列。我们将证明我们已经显着改进了制造工艺（目标 1），开发了一种宏孔成型器来生产 96 孔和 384 孔 CometChips（目标 2），并建议实施用于数据分析的图形用户界面（目标 3） ）。最后，我们将通过分析 NTP 库中一系列化合物的基因毒性效应来严格验证这项新技术，了解它们对 DNA 损伤和修复反应的影响，并揭示 DNA 损伤反应的个体间和细胞间类型变异（目标4）。通过整合生物学和工程学中的传统方法，这里描述的 DNA 芯片修复平台代表了一项重大的技术进步，提供了高通量、客观和定量的测量，有可能成为 DNA 损伤分析的新标准。