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）：保留基因组完整性对于抑制癌症，神经退行性，衰老和其他疾病至关重要。与基因组保存相反的是DNA损伤，它可以驱动突变，序列重排和细胞毒性。 DNA损伤是不可避免的，因为我们的环境和细胞中存在DNA损伤剂。为了抵消DNA损伤的有害影响，我们已经进化了复杂的DNA修复系统。现在众所周知，每种主要的DNA修复途径都会抑制癌症。此外，由于通常使用DNA损伤剂对癌症进行治疗，因此肿瘤的DNA修复能力调节对化学疗法的敏感性也就不足为奇了。尽管其重要性，但DNA损伤和修复的测量远非常规，这主要是由于缺乏可靠和快速的DNA损伤测定法。在这里，通过在工程师，生物学家和计算机程序员之间汇总融合专业知识，我们建议通过为快速半自动化的单细胞DNA损伤量化平台开发平台来满足这一需求，该平台可以广泛分发，并随时由公共卫生研究人员在公共卫生方面广泛应用， 学术，工业和医学。 如第一阶段提交中所定义的那样，我们为96孔Cometchip平台创建并测试了一个原型，并优化了工程设计和生产设备，以生成空间编码的20和96很好地证明，补充了Microwell Comet Gels，用细胞外基质蛋白（EMPS）的增长为启用了两个星期的人体和emps的生长。为了表征商业上使用的化学物质的遗传毒性，在环境或新开发的药物中发现的化学物质，并无需识别特定的DNA修复技术，可以量化DNA修复能力。该建议是为了开发芯片技术上的“ DNA修复”，结合了基于琼脂糖的微孔阵列，空间编码的细胞识别，自动数据处理以及细胞外基质蛋白来优化，验证，验证和商业化一系列空间编码的微孔阵列。我们将证明，我们已经显着提高了制造过程（AIM1），并开发了一个宏威的前者，以生产96孔和384-Welll cometchips（AIM 2），并提出实施图形用户界面以进行数据分析（AIM 3）。最后，我们将通过分析NTP库中一系列化合物的遗传毒性作用来严格验证这项新技术，从而影响了DNA损伤和修复反应，并揭示了DNA损伤反应中的个体间和细胞间型变化（AIM 4）。通过将传统方法整合在生物学和工程中，此处描述的芯片平台上的DNA修复代表了一项重大的技术进步，提供了高通量，客观和定量测量，具有成为DNA损伤分析中新标准的潜力。