(410c) Novel Approaches to High-Throughput Delivery of Silencing RNA

The development of genomic and proteomic technologies has led to the generation of enormous data sets of differentially expressed genes or proteins under varying conditions (e.g. environmental perturbations, cell differentiation, carcinogenesis, etc.) The critical issue is to identify which of these differentially expressed genes play a causal role in the various phenotypes observed under these conditions. One approach to identifying causality is the application of silencing RNA to reduce the activity of genes that are induced by the experimental system. However, silencing RNA does not readily lend itself to large-scale high-throughput analyses that would be compatible with analyzing hundreds of differentially expressed genes or proteins.

Using novel magnetic gene delivery reagents and viral transfection in combination with a proprietary three-dimensional (3D) cellular array chip (DataChip?), we have been able to transfect multiple genes and silencing RNA molecules into mammalian cells cultured in 3D arrays in 60 nL spots. Cell lines of clinical (MCF-7 breast cancer) and biotechnological (CHO-K1) relevance were transfected using this methodology. As proof of principle, cells were transfected with green fluorescent protein and the GFP was silenced using a shRNA against GFP. A variety of "toxic" shRNA molecules were also transfected and toxicity was verified by live-dead staining and analysis using a fluorescent microarray scanner. We have applied this system to investigate the responses of mammalian cells to hyperosmotic stress and evaluated the roles of five genes that were identified as differentially expressed in a microarray screen.

This system can be readily used to probe the roles of various genes in stem cell differentiation, responses to environmental stresses, or changes that occur during carcinogenesis, metastasis, etc. In addition, this system can be applied to knock down a wide range of pathways during in vitro analysis of potential pharmacological agents, permitting a mechanistic understanding of drug effectiveness.