The goal of this proposal is to develop a system for the localized, controlled, and efficient delivery of DNA from the cell culture substrate, which will transfect cells upon adhesion to the substrate. Efficient, controlled DNA delivery is a fundamental goal in biotechnology with applications to numerous therapeutic, diagnostic, and basic science applications. Substrate-mediated delivery provides a method to spatially control DNA delivery. The central hypothesis underlying this proposal is that DNA delivery to alter gene expression can be patterned through the tethering of DNA complexes to a cell adhesive substrate, which functions to retain the DNA in the cell microenvironment but allows for cell internalization. This strategy allows for DNA to be immobilized into patterns on the substrate, which will result in patterned gene expression within the cell population. This system will be developed to serve as a central technology for studies in tissue formation, and will enable numerous studies that are not currently possible. DNA is complexed with cationic polymers, a fraction of which contains functional groups for immobilization to the substrate. The tethering approach functions to ionically maintain the DNA at the substrate surface, yet allow for internalization.
The specific aims of the proposal will test the hypotheses that 1) cellular internalization of the DNA occurs by removal of the DNA from the cationic polymers that are tethered to the surface, and not the release of the complex into solution, 2) that optimal transfection results from a design that balances the need for cellular internalization with the need for substrate binding, and 3) patterned deposition of DNA encoding for neurotrophic factors can create patterns of of transfected cells that direct neurite outgrowth by primary embryonic neurons. Substrate-mediated delivery is a novel approach to DNA delivery that can be employed to spatially regulate gene transfer. Association of DNA with a substrate, which is an approach used by viruses, may enhance the in vivo applicability of non-viral DNA by preventing complex aggregation and facilitating cellular internalization. This system will provide the tools to recreate the complex patterns of gene expression that are observed within a developing tissue.
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