Real-time monitoring of gene expression by bioluminescence

基因表达分析对当今生物学的各个方面都至关重要。实时监测活细胞的基因表达是分析许多生物现象的分子机制的有力工具。为了将这项技术应用于更大范围的细胞，需要开发一种稳定和通用的方法。在这个项目中，我们分析了活体实时方法需要解决的许多技术问题。荧光素酶蛋白在细胞内的生物发光受到多种因素的影响，包括底物、酶的稳定性、能量供应等。为了避免这些因素，我们采用了两种铁道虫荧光素酶体系。这种昆虫有两个相似的荧光素酶基因。虽然两种荧光素酶具有相似的特性，但生物发光的波形却截然不同（540 nm和630 nm）。有了这些基因的供应，我们在不同的启动子下将它们引入蓝藻。在此基础上，研制了基于两根光电倍增管和不同波长干涉滤光片的生物发光自动检测系统。我们最终成功获得了含有两个荧光素酶报告子的蓝藻，并证实了两个启动子的信号可以被检测到而没有明显的串扰。因此，现在我们可以同时监测两个基因的表达。利用该方法，我们将提高生物发光信号的可靠性和更高的基因表达评估精度。该系统的应用可以扩展到各种生物系统，以获得可靠的基因表达监测。

Analysis of gene expression is essential for all aspects of biology today. Real-time monitoring of gene expression from living cells is very powerful tool to dissect molecular mechanisms for many biological phenomena. To apply this technique to wide range of cells, a stable and versatile method need to be developed. In this project, we analyzed many technical problem that the in vivo real-time method should address.Bioluminescence from luciferase protein in the cell is affected by various factors, including subtrate, stability of enzyme, energy supply, etc. To avoid these factors, we applied two luciferase system of railroad worm to this purpose. This insect have two similar luciferase genes. While two luciferases share similar characteristics, waveform of bioluminescence is quite different each other (540 and 630 nm). With suppy of thses genes, we introduce them to cyanobacteria under different promoters. Then we developed automated bioluminescence detection system based on two photomultiplier tube and interference filters of different wave length. We finally succeeded to obtain cyanobacteria that contains two luciferase reporter and confirmed that siginals from two promoters can be detected without significant cross-talk. Therefore, now we can monitor expressions of two gene concurrently. With this method, we will enhance reliability to bioluminescence signals and evaluate gene expression in higher precision. Application of these reproter system can be expanded to various biological system to obtain reliable monitor of gene expression.