LIGHT-ACTIVATED GENE EXPRESSION IN SINGLE CELLS (RMI)

DESCRIPTION (provided by applicant): This proposal is to develop 1) a photoactivatable gene that, upon exposure to light, begins transcription of visible nascent chains of RNA and 2) a protocol for subsequently detecting single RNA molecules in living cells and tissues. We will use the ecdysone response element and a caged, photoactivatable ecdysone. Into the gene we will insert an RNA reporter, containing 24 MS2 repeats, so expression can be monitored by the observation of a GFP-MS2 fusion protein that binds to the RNA transcripts. These RNAs have been detected as single molecules in cultured cells (Fusco et al., 2003). We intend to engineer this system into cancer cells that can be grown in culture and can form tumors in rats. Gene expression will then be initiated by uncaging the ecdysone in vivo first by conventional and then by two-photon microscopy. We will use these cells to form tumors in vivo and by intravital imaging (Condeelis and Segall, 2003), we will subject the tissue to a highly focused spot of photolysis through the microscope. In this way, the subsequent gene expression can be observed in a single cell within a tissue. The dynamics of single RNA molecule movements could then be described intravitally, and their distributions examined within a physiologically relevant system. The Specific Aims are: 1. Constructing a stable cell line with a photoactivatable gene and an mRNA reporter. 2. Photoactivating the gene and then detecting RNA transcription in real time and imaging the single RNA molecules released from the transcription site in single cells. 3. Intravitally imaging of this expression in a single cell within tumor tissue.