Method Development for a Next Generation Miniaturized Genomic Screening Platform

下一代小型化基因组筛选平台的方法开发

• 批准号: 8315375
• 负责人: Peter Kahn
• 金额: $ 14.98万
• 依托单位: ENGINEERING ARTS, LLC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8315375
• 关键词: AKT1 gene; Advanced Manufacturing Technology; Apoptosis; Arizona; Arts; Biological; Biological Assay; Biology; California; Cell Culture Techniques; Cell Death; Cells; Cessation of life; Chemicals; Computer software; Cultured Cells; Disease; Drops; Electroporation; Engineering; Enzymes; Equipment; Eukaryotic Cell; Family; Funding; G Protein-Coupled Receptor Genes; Gene Expression Regulation; Gene Family; Gene Proteins; Genes; Genome; Genomics; Goals; Grant; Hour; Human; Human Genome; Image Analysis; Individual; Institutes; Institution; Laboratories; Lead; Letters; Life; Liquid substance; Microarray Analysis; Microfluidics; Microscope; Microscopic; Molecular; Pathway interactions; Pharmacologic Substance; Phase; Phosphotransferases; Positioning Attribute; Printing; Process; Production; Protein Microchips; Public Health; Publications; Publishing; RNA Interference; RNA library; Reagent; Relative (related person); Research; Research Personnel; Running; Sampling; Screening procedure; Slide; Small Interfering RNA; Small RNA; Speed; Surface; TNFSF10 gene; Technology; Testing; Time; Universities; Vendor; base; biochip; caspase-8; commercial application; commercialization; cost; density; gene function; genome-wide; high throughput screening; innovation; insight; instrument; method development; miniaturize; nanolitre; next generation; novel; novel diagnostics; research study; response; small molecule; statistics; technological innovation; technology development

DESCRIPTION (provided by applicant): Every gene has its own set of small RNA molecules, known as small interfering RNA (siRNA), that inhibit expression of the gene's proteins. The siRNA molecules are part of an ancient natural mechanism of gene regulation, known as RNA interference (RNAi) that has been evolutionarily conserved since the earliest eukaryotic cells. More recently researchers have exploited RNAi to systematically knockdown, one-at-a-time, every single gene in living cell-cultures. These sets of experiments, known as cell-based genomic high throughput screening (HTS) provide insight into gene function especially as it relates to diseases and their treatments. Currently genomic HTS is typically done in 96 or 384-well microplates. A set of 100 plates is required to run HTS on the whole human genome. These large screens are now only done at well-funded institutions using rooms full of expensive automated liquid and microplate handling equipment. Our collaborator on this grant, Dr. Saez, has co-invented a novel HTS gene function platform, called, 'electroporation-ready- microwell-arrays', that will allow whole human genome screens on a single plate that is ready for cell culture and electroporation. The platform consists of a micro-machined array of electrically conductive micorwells that enable simultaneous electroporation of cultured-cells with thousands of different siRNA. This platform will enable genomic HTS to be routinely performed in smaller research labs which will dramatically increase the rate of discovery of new molecular pathways related to disease with corresponding impact on novel treatments and public health. As proof of concept, in this grant, we will perform a smaller screen for the human kinome, a set of 518 genes for kinase enzymes that are key controllers of cell activity and have great pharmaceutical significance. Engineering Arts has developed proprietary non-contact piezoelectric inkjet dispensing technology for microarraying applications. We have developed a high-speed microarraying instrument capable of on-the-fly dispensing of thousands of different sub-nanoliter sized reagents onto 36 individual microscope- slide substrates. Engineering Arts has already manufactured a handful of proof-of-concept electroporation- ready-microwell-arrays platforms for Saez's lab with encouraging preliminary results. Under this grant we will develop the additional manufacturing technology required to automatically align the relative positions of the microscopic features of Saez's platform within +/- 20 um. We will also increase high-speed dispensing volume to ~10 nanoliter per microwell. Together these innovations will allow manufacturing production rates of 36 electroporation-ready-microwell-arrays platforms in 8 hours. After manufacturing the platforms, they will be tested in Saez's lab at the SCRIPPS Institute in La Jolla California and Pedro Aza's lab at Burnham, who ran a similar screen using conventional 384-well plates. The novel alignment technology and dispensing technology developed under this grant will enable many other biomedical applications that require the capability to deliver small fluid volumes precisely to microscopic features on 'biochips'. PUBLIC HEALTH RELEVANCE: New miniature test platforms are being developed with thousands of microscopic features to do thousands of biology experiments simultaneously. In this grant, Engineering Arts will develop the critical manufacturing technology to rapidly and cost-effectively deliver thousands of different nanoliter-sized drops of biological molecules or other chemicals to those features. The value of the manufacturing technology will be demonstrated on a platform that enables the simultaneous study of the function of thousands of genes in living cells.

描述(由申请人提供)：每个基因都有自己的一组小RNA分子，称为小干扰RNA(SiRNA)，它们抑制基因蛋白质的表达。SiRNA分子是一种古老的自然基因调控机制的一部分，称为RNA干扰(RNAi)，自最早的真核细胞以来一直在进化上保守。最近，研究人员利用RNAi系统地一次一个地敲除活细胞培养中的每一个基因。这些实验被称为基于细胞的基因组高通量筛选(HTS)，它提供了对基因功能的洞察，特别是当它与疾病及其治疗相关时。目前，基因组高温超导通常是在96或384孔微孔板中完成的。在整个人类基因组上运行HTS需要一套100个平板。这些大屏幕现在只在资金充足的机构使用满是昂贵的自动化液体和微板处理设备的房间进行。我们在这笔赠款上的合作者赛兹博士与人共同发明了一种新的HTS基因功能平台，名为“电穿孔就绪微孔阵列”，它将允许在单个平板上进行全人类基因组筛选，为细胞培养和电穿孔做好准备。该平台由微机械加工的导电微孔阵列组成，能够同时电穿孔数千种不同的siRNA培养细胞。这一平台将使基因组HTS能够在较小的研究实验室中常规进行，这将极大地提高与疾病相关的新分子途径的发现速度，并对新的治疗方法和公共健康产生相应的影响。作为概念的证明，在这项拨款中，我们将对人类基因组进行较小的筛选，这是一组518个激酶酶基因，是细胞活动的关键控制因素，具有重大的药学意义。工程艺术公司为微阵列应用开发了专有的非接触式压电喷墨点胶技术。我们开发了一种高速微阵列仪器，能够将数千种不同亚纳升大小的试剂即时分配到36个单独的显微镜载玻片基板上。工程艺术公司已经为赛兹的实验室制造了几个概念验证电穿孔准备微孔阵列平台，取得了令人鼓舞的初步结果。根据这笔赠款，我们将开发所需的额外制造技术，以在+/-20um内自动对准赛斯平台的微观特征的相对位置。我们还将把高速点胶量增加到每微孔~10纳升。这些创新加在一起，将使36个电穿孔准备就绪的微孔阵列平台的生产速度在8小时内达到。制造完平台后，它们将在加利福尼亚州拉霍亚的斯克里普斯研究所的赛斯实验室和伯纳姆的佩德罗·阿扎的实验室进行测试，后者使用传统的384孔板运行类似的屏幕。根据这笔赠款开发的新的对准技术和分配技术将使许多其他生物医学应用成为可能，这些应用需要将小体积的液体精确地输送到“生物芯片”上的微观特征。 与公共健康相关：新的微型测试平台正在开发中，具有数千个微观特征，可以同时进行数千个生物实验。在这笔赠款中，工程艺术公司将开发关键的制造技术，以快速且经济高效地为这些功能提供数千个不同纳升大小的生物分子或其他化学物质。制造技术的价值将在一个能够同时研究活细胞中数千个基因功能的平台上得到展示。