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Molecular, Biochemical, and Genetic Techniques

開催日 2014/9/12
時間 14:00 - 15:00
会場 Poster / Exhibition(Event Hall B)

Improved bioluminescent reporters based on newly cloned firefly luciferases

  • P2-380
  • 杉山 崇 / Takashi Sugiyama:1 
  • 1:オリンパス株式会社 / Olympus Corporation, Tokyo, Japan 

Bioluminescent imaging is a powerful tool for investigating intracellular signaling. Previously, we demonstrated that human opsin5 (OPN5) photoreceptor mediates light-induced Ca2+ response in human embryonic kidney (HEK293) and mouse neuroblastoma (Neuro2a) cell lines using a luminescence imaging system with a synthesized bioluminescent indicator based on commercially available firefly luciferase (Luc2; Promega). Although this probe allow for live-cell imaging of a receptor-mediated slow Ca2+ response, bioluminescence signals were too weak to use for a high-speed imaging. To overcome this problem, we chose the brighter firefly luciferases derived from Pyrocoelia matsumurai (OKI), Drilaster kumejimensis (KUME), and Stenocladius flavipennis (SfRE1) to make the brighter Ca2+ indicators. First, we made circularly permutated luciferases (CPLs) of these firefly luciferases, because the Ca2+ indicator based on CPL had a high signal-to-noise ratio and a wide dynamic range. Among these CPLs, we obtained several brighter CPLs derived from OKI luciferase. The newly synthesized CPLs showed 2- to 3-fold brighter luminescence than Luc2-based CPL. Moreover, we produced the heterogeneously fused CPLs and demonstrated that these CPLs have 3- to 10-fold brighter luminescence than Luc2-based CPL. Using these CPLs, we successfully synthesized bioluminescent Ca2+ indicators with a bright luminescence and a wide dynamic range in signal change. The newly synthesized bioluminescent indicator allowed acquisition with a sampling rate as fast as 5Hz from single cell level and visualization of serotonin-induced Ca2+ oscillation in HEK293 cells expressing serotonin 2A receptor.
Furthermore, we made a reporter system for gene expression using a strategy based on the complementation of split luciferase of these luciferases. We designed the split sites of the luciferases and successfully developed the split luciferase reporters with intensity bright enough for imaging. Using these reporters, we obtained a time-lapse imaging of the c-fos promoter activity in HEK293 cells expressing human D1 dopamine receptor. Combination with a cell-specific expression technique, we are trying to develop a system for visualization of cell-specific gene expression.

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