Brighter reporter genes from multimerized fluorescent proteins.

814 BioTechniques Vol. 39, No. 6 (2005) The discovery and development of the green fluorescent protein (GFP) has opened up new areas of biological research, particularly for the visualization of protein dynamics and gene expression in living cells and organisms (1,2). The development of wavelengthshifted variants of GFP has further expanded the impact on biological research. One of the fluorescent proteins’ most important applications has been as a tag for protein localization and turnover. Although attempts have been made to use XFPs [cyan fluorescent protein (CFP), GFP, and yellow fluorescent protein (YFP)] as reporters of promoter activity, these efforts have been less successful primarily because most promoters are not active enough to yield sufficient fluorescent protein accumulation to visualize without antibody enhancement (3–5). Nevertheless, almost all applications can be enhanced by improving the spectral yield of fluorescent proteins, which therefore has become an important research goal. For example, enhanced GFP (EGFP) (6) and Venus (7) have become widely used following their introduction, such that EGFP is currently the standard fluorescent protein used in biological applications. Despite these developments, detection problems still remain an issue, especially when XFPs are used to report promoter activity in vivo in tissues from large multicellular organisms such as the mammalian central nervous system (CNS) (4,5). This is true whether fluorescent proteins are used as reporter genes directly following the promoter as well as when they are expressed as bicistronic messages following an internal ribosome entry site (IRES) sequence (8). In the latter case, gene expression downstream of the IRES is diminished, and fluorescence output is decreased or even absent in these constructs compared with that found in fusion proteins (9,10). Thus, improved fluorescent protein spectral yield will open up new possibilities for reporting spatial and temporal analysis of vertebrate gene expression in vivo. As an attempt to improve the spectral yield of XFP fusion proteins, some investigations have employed tagging proteins of interest using multiple XFPs linked in tandem (11–13). However, despite the qualitative success of this approach for particular fusion proteins, no quantitative analysis of the increase in fluorescence output or the limits of this approach after multimerization has been performed. This work sought to quantitatively determine the improvements in spectral yield after XFP multimerization, with a particular emphasis on using these constructs as reporters of gene expression. Here we report quantitative and significant increases in fluorescence output after in-frame multimerization of XFP monomers. This enhancement is observed using a range of multimerized fluorescent proteins, effectively boosts spectral yield in IRES expression constructs, and can be employed using mammalian Brighter reporter genes from multimerized fluorescent proteins