DETECTING MRNAS IN LIVING CELLS WITH MOLECULAR BEACONS

目前正在开发一种可视化活细胞中特定mRNA序列的技术。该技术利用分子信标，这是一种寡核苷酸探针，在杂交后变成荧光。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，并以细胞为基础对基因表达进行成像。通过使用黑腹果蝇合胞胚胎的模型系统，我们将展示在活细胞中可以实时观察到不同mrna定位发生的动态变化。通过引入几种不同分子信标的混合物来绘制同一细胞内多个mRNA的分布，其中每个分子信标对不同的mRNA靶标具有特异性，并且每个分子信标用不同颜色的荧光团标记。通过将分子信标微注射到原代培养的神经元核周空间中，可以绘制一组mrna的分布，这些mrna在突触后树突位置响应突触刺激。利用共聚焦激光扫描显微镜监测mRNA在神经元内以核糖核蛋白颗粒形式的细胞内转运，以阐明mRNA定位的机制。将开发将分子信标有效递送到悬浮细胞中的技术，并使用荧光激活细胞分选技术监测单个细胞中的基因表达。利用分子信标检测活细胞中mRNA的能力将使我们能够以比以前更清晰的焦点探索神经元和其他细胞内mRNA定位和运输的分子复杂性。

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.