Flow Cytometric Analysis of Gene Function

-HTP siRNA screening assay by using Fluorescent Proteins-

Abstract

Gene silencing is widely used to clarify the gene function. Although RNA interference (RNAi) can expect a handy and large effect for gene silencing, the development of the screening system of a target gene is important. Green fluorescent proteins (GFPs) and Reef Coral fluorescent proteins (RCFPs) are well known that can make stable fluoresce without any co-factors in almost all cells. Both of the function of the target gene and the fluorescent of GFPs/RCFPs are most probably kept, even if a lot of genes are made as a fusion protein with these fluorescent proteins. When GFP or RCFP is connected with C-terminal of a target protein, and if a target protein receives the resolution by RNAi, the fluorescence of GFP or RCFP in the downstream of a target protein cannot be translated to a protein and fluorescence disappears. We developed the high throughput screening system for RNAi. Fluorescence-Activated Cell Sorter (FACS) measurement of the fluorescence of 293 cells after the expression vector of a target gene and RNAi vector were co-transfected to 293 cells, a handy screening of RNAi can be established.