This Small Business Innovation Research Phase I project aims to develop a novel reporter gene assay for studying eukaryotic gene expression and regulation in individual transfected cells, and for isolating variants with defined transcription responses for cloning and use in subsequent studies. This assay will use single cell gel microdrop technology, a secreted enzyme capture and detection format, and fluorescence activated cell sorting. Although there have been significant developments in reporter gene assays, an assay which provides high sensitivity, high throughput and single cell resolution is still needed. Frequently, gene products may be difficult to detect due to low abundance or instability. In addition, many potential therapeutic molecules that modulate transcription, exhibit weak target binding affinities, resulting in low reporter gene expression. The proposed assay will permit detection of a wide range of signal intensities from single, transfected cells which can be isolated for subsequent cloning. Any study of a biochemical process that terminates in a transcriptional event would benefit from use of the proposed reporter gene assay. This assay will facilitate drug discovery by permitting rapid screening of combinatorial chemistry or natural product libraries.
