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Abstract
Grant Number: 1P20EB004930-01 Project Title: LIGHT-ACTIVATED GENE EXPRESSION IN SINGLE CELLS (RMI)
PI Information: Name Title SINGER, ROBERT H. rhsinger@aecom.yu.edu PROFESSOR AND CO-CHAIR Abstract: 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.
Public Health Relevance:
This Public Health Relevance is not available.Thesaurus Terms:
ecdysone, gene expression, genetic regulation, messenger RNA, photoactivation, technology /technique development
cell line, chimeric protein, genetic regulatory element, genetic transcription, photolysis, reporter gene
green fluorescent protein, laboratory rat
Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2004 Department: ANATOMY AND STRUCTURAL BIOLOGY Project Start: 01-AUG-2004 Project End: 31-JUL-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZGM1