Vol.1 No.4 2009

18/79

Research paper : From basic research on firefly bioluminescence to Product Realization Research (Y. Ohmiya et al.)−235 Synthesiology - English edition Vol.1 No.4 (2009) Ltd., and “MultiColor Luc” from Toyo B-Net Co., Ltd. We have sold the kits from these companies, as we had projected as an outcome of the proposal. The decision whether the company will sell the product depends on the demand for the products and the number of users and potential users that demand improvement in technology and expect the introduction of new technology. Japanese bioresearchers tend to take up technology they see in foreign journals rather than using their own judgment to introduce and adopt new technology. It is important for researchers to be alert to new developments and for those who produce new biotechnology to communicate the advantages of their products through as many channels as possible. Conducting Type 2 Basic Research and practical research that starts from a patent will not lead to true product realization unless it is followed up by continued aftercare, which produces solid results from Type 1 Basic Research based on the new technology created.The labeling technology for studying multiple types of biomolecules involves monitoring the expression of several genes in the cell. The expression of several genes is regulated by quick or slow responses to external stimulus in the cell, which produces various proteins. For example, when an environmental hormone reaches the cell, the cell produces a female hormone in response. To detect the expression of a certain gene, we developed a reporter assay using the firefly bioluminescence enzyme, luciferase. In this reporter assay, the promoter region of the gene sequence that regulates the expression of the gene is inserted into the firefly luciferase gene and this is then transduced into the cell. If the gene expression is induced, luciferase is synthesized accordingly. Because luminescence occurs when luciferin is added to the expressed luciferase, the extent of genetic expression can be assessed by the amount of luminescence. This method was already employed in the biomedical field and has become a standard chemical assay in the environmental field (approved as an official method by the Ministry of Environment) and in screening methods for drug discovery. In 2002, the method was part of a 500 million-yen market in Japan and 20 billion-yen market worldwide.Is it possible that these companies will realize their products in this field? The reporter assay that was supported widely until 2002 was the dual reporter sold by Company P. This product used a two-step measurement method comprising two genes and two substrates, and Company P, which held the patent in the United States, dominated the market. Companies trying to break this domination had to search for other potential reporter assay systems. Users were dissatisfied because the method could assess expression of two genes only, and there were problems relating to the cost and the two-substrate two-step process. Potential users wanted to a reporter assay whose application could be extended to more genes. We were able to set a clear goal to develop a new “three-genes, one-substrate one-step reporter assay” using the principle of the diversity of luminescence color difference.3 Patent construction for product realization from Type 2 Basic ResearchThe light emitted by the firefly occurs through an enzymatic reaction in which the oxidation of firefly luciferin is catalyzed by firefly luciferase. There families of luminescent beetles are the Lampyridae (firefly), Elateridae, Drilidae, and Rhagophthalmidae (Iriomote firefly) families. These are unique in that their colors of luminescence differ slightly and because the luciferin–luciferase reaction color may be changed depending on the pH of the reaction environment. We decided to detect the multiple gene expression using luciferases that produced different colors but that were not influenced by a pH change. We have already isolated the luciferase genes that produce red and green light from the South American railroad worm, which produces varied luminescence (i.e., the head emits orange to crimson colors while the abdomen emits green to yellow-green colors). We have succeeded in expressing luciferase in E. coli and in producing orange luminescence using this genetic information[3][4].However, when applying the multiple gene expression detection kit in 2002, we were unable to produce the stable expression of luciferase in mammalian cells. Although we (15)−Fig.3 Outline of technology for dividing and measuring three luminescence colors using two filters.A) Luminescent spectrum group of green, orange, and red luciferases.B) Optical absorbance of two filters used and luminescence spectra when three colors are mixed.C) F0 is the amount of light when all-optical output is measured without a filter.D) Most green light is absorbed when passing through the first filter. F1 is the amount of light not measured.E) Most green and orange light is absorbed when passing through the second filter. F2 is the amount of light not measured.F) Equation to calculate the original amount of luminescence by calculating F0, F1, F2, and the transmission coefficient of the two optical filters, which had been predetermined.Relative light intensityTransmission rate (%)Wavelength (nm)ABCDEF400 500 600 7001.00.80.60.40.201.00.80.60.40.20010080604020Transmission rate (%)Wavelength (nm)400 500 600 700010080604020Relative light intensityF0F0F1F1F2F2=F0F1F2〔 〕GOR〔 〕1 1 1KGO56KOO56KRO56KGO60KOO60KRO60〔 〕

※このページを正しく表示するにはFlashPlayer9以上が必要です