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

我们开发了一个系统，在该系统中，单个细胞中的多个基因表达被记录为不同波长的生物发光。该方法适用于活细胞中分子的实时分析。灌注培养系统的开发：为了监测培养细胞的特定基因的表达，我们开发了一种灌注系统，在该系统中连续测量报告活性。在这项研究中，我们使用了一种分泌型荧光素酶（vargula hilgendorfii），其DNA连接到生长激素（GH）启动子的下游。灌注培养液中荧光素酶蛋白的含量与生物发光呈明显的正相关，证明了生物发光测定的可靠性。我们还通过使用转录、翻译或分泌囊泡释放的特异性阻断剂来检测报告荧光素酶的合成、胞内转运和分泌的时间过程.基因监测时钟的构建 ...更多信息 载体：我们构建了一个载体，其中mPer 1启动子区8.1kb连接到萤火虫荧光素酶DNA的上游。将它们转染到NIH 3 T3细胞系中，获得稳定的转化子。通过加入CLOCK/BMAL 1表达载体并观察生物发光的增加来评价报告系统.高灵敏度发光检测装置的研制：1）一种新的发光绝对值检测方法（1）用激光获得了发光相对强度向绝对值的转换因子。(2)使用获得的转换因子校准荧光素酶的标准溶液。(3)2）多色活细胞荧光检测装置的研制我们研制了一种检测系统，该系统利用特定的滤光片和高强度的CCD摄像机同时监测多种不同波长的生物荧光。少

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