Method Development for a Next Generation Miniaturized Genomic Screening Platform

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)，可以抑制基因蛋白质的表达。siRNA分子是一种古老的基因调控自然机制的一部分，被称为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），提供了对基因功能的深入了解，特别是当它与疾病及其治疗有关时。目前，基因组HTS通常在96孔或384孔微孔板上进行。在整个人类基因组上运行HTS需要一组100个板。这些大屏幕现在只能在资金充足的机构中使用充满昂贵的自动化液体和微孔板处理设备的房间来完成。我们的合作者，Saez博士，共同发明了一种新的HTS基因功能平台，称为“电穿孔-微孔阵列”，它将允许在单个板上进行整个人类基因组筛选，准备进行细胞培养和电穿孔。该平台由一个微机械阵列的导电微孔组成，可以同时用数千种不同的siRNA对培养细胞进行电穿孔。该平台将使基因组HTS能够在较小的研究实验室中常规进行，这将大大提高发现与疾病有关的新分子途径的速度，并对新的治疗方法和公共卫生产生相应的影响。作为概念验证，在这项拨款中，我们将对人类kinome进行更小的筛选，kinome是一组518个激酶基因，是细胞活性的关键控制因子，具有重要的药物意义。工程艺术开发了专有的非接触式压电喷墨点胶技术，用于微阵列应用。我们开发了一种高速微阵列仪器，能够在36个单独的显微镜载玻片上实时分配数千种不同的亚纳升大小的试剂。Engineering Arts已经为Saez的实验室制造了一些概念验证的电穿孔微孔阵列平台，并取得了令人鼓舞的初步结果。根据这笔资金，我们将开发额外的制造技术，以在+/- 20um内自动对齐Saez平台的微观特征的相对位置。我们还将增加高速分配容量至每微孔约10纳升。这些创新将使36个电穿孔微孔阵列平台的生产速度在8小时内实现。制造出平台后，它们将在加利福尼亚州拉霍亚的斯克利普斯研究所的Saez实验室和伯纳姆的Pedro Aza实验室进行测试，后者使用传统的384孔板进行类似的筛管。在这项资助下开发的新型对准技术和分配技术将使许多其他生物医学应用能够将小体积流体精确地输送到“生物芯片”的微观特征上。

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.