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Brighter reporter genes from multimerized fluorescent proteins.
If low signal-to-noise ratios in GFP assays have you singing a sad tune, Genové et al. (p. 814) can offer a solution. Recognizing that GFP fluorescence can be almost undetectable when reporters are used downstream of relatively weak promoters or during detection in problematic tissues such as the CNS, the authors investigated whether tandem repeats of fluorescent proteins could yield quantitative data in reporter gene assays. In search of the best readout, Genové et al. compared fluorescence intensity of one, two, or three copies of EGFP, EYFP, and Venus. Although a significant improvement in detection was evidenced with the trimeric EGFP, six tandem copies revealed fluorescence levels similar to the one-copy construct. Trimeric EYFP showed a similar improvement in signal, as both an IRES-translated protein and as a protein fusion with Fos. However, the brightest results involved Venus, a highly optimized EYFP variant. The trimeric Venus construct displayed a 12-fold higher fluorescence output than a single EFYP, and even after 5 minutes of constant illumination remains severalfold brighter. Based on this work, the prospects of previously problematic reporter assays should brighten considerably.