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.
