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.
|Effective start/end date||9/30/99 → 9/29/03|
- National Institutes of Health: $664,784.00
- National Institutes of Health
- National Institutes of Health: $473,423.00
- Environmental Science(all)