DETECTING MRNAS IN LIVING CELLS WITH MOLECULAR BEACONS

目前正在开发一种技术，用于显示活细胞中特定的mRNA序列。这项技术利用了分子信标，这是一种寡核苷酸探针，在杂交时会变成荧光。使用分子信标可以同时绘制活细胞内多个mRNA的细胞内分布图，并在逐个细胞的基础上对基因表达进行成像。使用果蝇合胞胚胎的模型系统，我们将展示在活细胞中实时观察不同mRNAs定位的动态变化。通过引入几个不同分子信标的混合物，将在同一细胞内映射多个mRNAs的分布，其中每个分子信标特定于不同的mRNA靶标，每个分子信标被标记有不同颜色的荧光团。通过将分子信标微量注射到原代培养神经元的核周空间，将定位于突触后树突状细胞位置的一组mRNAs的分布将被映射。为了阐明mRNAs定位的机制，将使用共聚焦激光扫描显微镜监测核糖核蛋白颗粒形式的mRNAs在神经元内的细胞内运输。将开发有效地将分子信标输送到悬浮细胞中的技术，并将使用荧光激活的细胞分选来监测单个细胞中的基因表达。通过分子信标在活细胞中检测mRNAs的能力将使我们能够探索mRNA在神经元和其他细胞内定位和运输的分子复杂性，其焦点比以往任何时候都更清晰。

A technique is being developed for the visualization of specific mRNA sequences in living cells. This technique utilizes molecular beacons, which are oligonucleotide probes that become fluorescent upon hybridization. Use of molecular beacons allows the simultaneous mapping of the intracellular distribution of multiple mRNAs within living cells and imaging of gene expression on a cell-by-cell basis. Using a model system that employs syncytial embryos of Drosophila melanogaster, we will show that the dynamic changes that occur in the localization of different mRNAs can be observed in real-time in living cells. The distribution of multiple mRNAs will be mapped within the same cell by introducing a mixture of several different molecular beacons, where each molecular beacon is specific for a different mRNA target, and each molecular beacon is labeled with a differently colored fluorophore. The distribution of a group of mRNAs that localize at postsynaptic dendritic sites in response to the synaptic stimuli will be mapped by microinjecting molecular beacons into the perinuclear space of neurons maintained in primary cultures. The intracellular transport of mRNAs within neurons in the form of ribonucleoprotein particles will be monitored using confocal laser scanning microscopy, in order to shed light on the mechanism of mRNA localization. Techniques will be developed for an effective delivery of molecular beacons into cells in suspension and gene expression will be monitored in individual cells using fluorescence-activated cell sorting. The ability to detect mRNAs in living cells with molecular beacons will allow us to explore the molecular complexities of mRNA localization and transport within neurons and other cells with a sharper focus than has been possible before.