Development of multidimensional real-time monitoring system for neuronal activity by plural bioluminescent reporters

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We developed a system in which multiple gene expressions in a single cell were recorded as bioluminescence of different wave lengths. The method is applied for real-time analyzes of molecules in living cells.1. Development of a perfusion culture system : In order to monitor the expression of a particular gene of cultured cells, we developed a perfusion system in which reporter activity was continuously measured. In this study, we used a luciferase of a secretion type (vargula hilgendorfii), the DNA of which was connected with the downstream of growth hormone (GH) promoter. The amount of luciferase protein in the perfusion culture medium and bioluminescence showed a clear positive correlation and proved a reliability of bioluminescence measurement. We also examined the time course of synthesis, intracellular transport and secretion of reporter luciferase by using specific blockers for transcription, translation or release of secretion vesicles.2. Construction of a clock gene monitoring … More vector : We constructed a vector in which a mPer1 promoter region of 8.1kb was connected to the upstream of firefly luciferase DNA. They were transfected to the NIH3T3 cell line and stable transformants were obtained. The reporter system was evaluated by adding CLOCK/BMAL1 expression vectors and observing the increase of bioluminescence.3. Development of a luminescence detection apparatus with high sensitivity :1) A novel method for detecting absolute levels of luminescence(1) A factor for transformation of relative intensity of luminescence to an absolute value was obtained by laser.(2) The standardized solution of luciferase was calibrated using an obtained transform factor.(3) The unknown sample was quantified its reporter activity by the above mention standard curve.2) Development of multi-color detection apparatus for living cellsWe developed a detection system in which more than one bioluminescence of different wave lengths were monitored simultaneously using specific filters and intensive CCD camera. Less