There is an increasing demand for high throughput (HTP) methods for gene 
analysis on a genome-wide scale. However, the current repertoire of HTP 
detection methodologies allows only a limited range of cellular phenotypes to be 
studied. We have constructed two HTP-optimized expression vectors generated from 
the red fluorescent reporter protein (RFP) gene. These vectors produce 
RFP-tagged target proteins in a multiple expression system using gateway cloning 
technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing 
us localize the expressed proteins in mammalian cells. The effectiveness of the 
vectors was evaluated using an HTP-screening system. Sixty representative human 
C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor 
cells, and we studied in detail two C2 domains that promoted the neuronal 
differentiation of HiB5 cells. Our results show that the two vectors developed 
in this study are useful for functional gene analysis using an HTP-screening 
system on a genome-wide scale.