[
International Worm Meeting,
2005]
Fluorescent Timer, a proprietary fluorescent reporter from Clontech, has the unique property of shifting its emission fluorescence from green (480nm) to red (583nm) in a time/intramolecular reaction dependent manner. Red fluorescence was documented to occur two to four hours after initial green fluorescence is detected (Terskikh et al. 2000). I have attempted to use this reporter to assay for message turnover under the control of various upstream regulatory regions. Regulatory regions were selected on the basis of showing limited temporal expression during early development and/or in various tissues to test the efficacy of the reporter as a general assay of transcriptional regulation. Regulatory regions were linked to the reporter via PCR fusion (Hobert, O. 2002). All transgenic lines containing upstream regulatory regions fused to the raw reporter failed to produce fluorescence. Upon the addition of a 3UTR and the insertion of two adenosine residues directly upstream of the translational start site, 10/14 transgenic lines displayed fluorescence. Embryonic expression, which was observed using the same upstream regulatory regions fused to GFP, was not observed in any of the Fluorescent Timer reporter lines constructed. To possibly counter to effects of cytoplasmic toxicity of the Fluorescent Timer protein, and to concentrate the signal to the nucleus, I fused a 5NLS to the construct. Unexpectedly, only 3/14 lines displayed fluorescence leading to the assumption that Fluorescent Timer may be more toxic when concentrated to the nucleus. This result seems to agree with the results that no Fluorescent Timer expression was observed during early development possibly due to toxicity of the construct. Fluorescent Timer may be a useful system for the assay of temporal regulation of transcription during later stages of C. elegans development. References: Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, Zhao X, Lukyanov S, Matz M, Kim S, Weissman I, Siebert P. 2000, "Fluorescent timer": protein that changes color with time. Science. Nov 24;290(5496):1585-8. Hobert O. 2002, PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques. Apr;32(4):728-30.