Final Report Summary - GENEXP (Gene Expression Explored in Space and Time Using Single Gene and Single Molecule Analysis)
Live-cell imaging combined with kinetic analyses has provided new biological insights on the gene expression pathway. However, such studies in human cells typically require use of exogenous over-expressed gene constructs that usually lack the complete endogenous regulatory sequences. It was therefore imperative to design methodology for analysing gene expression kinetics of single alleles of endogenous genes. While certain steps were previously taken in this direction, there have been many experimental obstacles standing in the way of a robust genome-wide system for the in vivo examination of endogenous gene expression within the natural nuclear environment. In this project we established an approach by which we tag endogenous genes in mammalian cells on the RNA and protein levels, simultaneously. The human cell clone library of such endogenous tagged genes that we accumulated is being used for the imaging of gene expression of these single genes. To follow the next step of the pathway, namely, the travels of the mRNAs from the nucleus to the cytoplasm, we have established a FRET-FLIM system that allows us to identify the specific interaction of mRNA export factors with different components of the nuclear pore complex (the gates of the nucleus), on the level of individual cells. Finally, we performed high-content screening of small molecule libraries in order to detect new compounds (therapeutics) that will interfere with the gene expression pathways we are studying, and thereby to identify new ways by which to manipulate the activity of genes and affect diseased states.