Multichannel analysis and monitoring of multiple gene expressions by using bioluminescent reporters

通过导入编码荧光素酶不同波长发光的报告基因，我们监测了多个基因在单个细胞中的表达。该方法被应用于二维和时程分析，以可视化活细胞内分子的相互作用。(1)新的生物发光报告基因的克隆和多色报告载体的构建：铁路蠕虫的两个荧光素酶通过催化同一底物发出红光和绿光。通过插入加压素和CMV启动子下的cDNA构建了双报告载体。虽然可以检测到发光，但哺乳动物细胞中的发光很弱。因此，利用海参荧光素酶和萤火虫荧光素酶构建了强度更高的双报告基因。将生长激素(GH)基因的启动子序列连接到vargula荧光素酶基因的上游，并将报告载体导入GH3细胞。The St…进一步转导萤火虫荧光素酶，可获得较多的GH3细胞集落。多色监测系统的构建：我们构建了由37℃暗盒组成的检测系统，暗盒中装有6个草底培养皿的转盘在冷却的LCD摄像机上旋转。通过液晶滤光片分离峰值波长的光子数。(3)活细胞中启动子序列的克隆和靶基因表达的连续监测：利用与绿色荧光蛋白或萤火虫荧光素酶基因相关的不同长度的小鼠PER1和BMAL1基因的启动子序列来分析转录调控。我们确定了启动子中的调控区域，并构建了双色报告基因来监测PER1和BMAL1的表达。我们正在构建转基因小鼠品系，以研究监控这两个时钟基因表达的分子时钟机制。较少

By transfecting reporter genes which encode luciferase emitting light with different wave lengths, we monitored expression of multiple genes in single cells. The method was applied for two dimensional and time-course analyzes to visualize the interactions of molecules within living cells.(1) Cloning of novel bioluminescence reporter genes and construction of multi-color reporter vector : Two luciferases of the railroad-worm emit red and green lights by catalyzing the same substrate. The dual reporter vector was constructed by inserting the cDNAs, one under the vasopressin and the other under CMV promoter. Although the light emission could be detected, the luminescence in the mammalian cell was weak. Therefore, dual reporter with higher intensity was constructed using vargula hilgendorfii luciferase and firefly luciferase. Promoter sequence of growth hormone (GH) gene was connected to the upstream of vargula luciferase cDNA and the reporter vector was transfected to the GH3 cell. The st … More able colony of GH3 cells was further transfected firefly luciferase. Luminescence from vargula and firefly luciferases was separately monitored from GH3 cells continuously.(2) Construction of multi-color monitoring system : We constructed the detection system consisting of dark box kept 37 ℃ in which a turn table carrying 6 grass-bottom petri dishes rotates on the cooled CCDcamera. A luminocapter counted the photons after separating those with the peak wavelength through liquid crystal filters.(3) Cloning of promoter sequences and continuous monitoring of targeted gene expression in living cells : Transcriptional regulation was analyzed using various length of promoter sequences of mouse Per1 and BMAL1 genes connected with either GFP or firefly luciferase cDNA. We identify the regulatory region in the promoter and constructed dual color reporter to monitor Per1 and BMAL1 expressions. We are constructing the transgenic mice line to study the molecular clock mechanisms monitoring the expression of the two clock genes. Less