Research Project
6833343
DESCRIPTION (provided by applicant): RNA interference (RNAi), a specific gene silencing method mediated by an intracellular enzyme complex using a dsRNA template, is of great current interest due to important advantages over older antisense methods. Recent reports have touted the utility of RNAi for basic and applied research aimed at determining functions of gene products and validating drug targets, as well as for therapeutic approaches. However, because nucleic acids do not readily pass through living mammalian cell membranes, robust techniques to deliver reagents into target cells are needed to fully realize the potential of RNAi-mediated gene silencing. We have developed a novel laser-based cell processing system, called LEAP (Laser-Enabled Analysis and Processing), for high-speed cell imaging and laser-based manipulations. LEAP images cells at >10[5] per second, and laser-irradiates specific cells at >10[3] per second leading to various cell manipulations such as cell death (i.e., for cell purification) or optoinjection (i.e., reversible permeabilization of cells for transfection). During Phase I, LEAP was used to demonstrate successful optoinjection of RNA- and DNA-based reagents into cells for achieving RNAi. Importantly, optoinjection compared favorably to commonly-used lipid-based transfection methods with respect to cell viability and transfection efficiency. The overall goal of Phase II is now to significantly advance this capability and to demonstrate the use of LEAP for enabling large-scale RNAi-based functional genomics studies. Specifically, the Phase II aims are to: (i) continue optimization of optoinjection for RNAi including difficult cell types; (ii) evaluate impact of optoinjection on cell physiology and compare to existing methods; and (iii) implement functional genomics assays on LEAP. Phase II will demonstrate broad utility of LEAP for in situ gene silencing by optoinjection of siRNA in a variety of cell types to affect a variety of cellular processes. The specific benefits (i.e., pros and cons) of optoinjection versus other transfection methods will be established through a variety of approaches to demonstrate the physiological impact on cells undergoing transfection. The resulting data will be of broad interest and will benefit the scientific community by evaluating cell responses following the use of various transfection methods. With such data from Phase II, a Phase III commercialization effort would be enabled to promote adoption of the optoinjection approach to realize the potential that RNAi technology holds for advancing functional genomics efforts.
