SINGLE MOLECULE DYNAMICS OF GENE EXPRESSION MEASURED ON SINGLE GENES IN LIVING CELLS

How can the transcriptional output of a gene be measured in a living cell? One approach is to measure transcriptional kinetics on multiple-copy gene-arrays by quantifying the rates at which fluorescently tagged mRNA is produced. This technique can provide accurate rates of transcription elongation in vivo. Another system allows the detection of transcriptional gene activity from a single gene, thereby providing the ability to analyze the kinetics of gene expression on single alleles. We use such analysis combing high resolution 3D RNA FISH on the single molecule level together with quantitative 4D live-cell imaging to quantify gene expression in single cells. We are able to discern between endogenous and over-expressed states of a gene, to provide spatial and temporal information on transcription throughout the cell cycle, to examine the effect of chromatin modifications on the transcriptional output, and to follow the signal transduction pathway and signaling proteins all the way from the cell membrane to the gene promoter. Thereby we can measure and demonstrate the modulation of the transcriptional output in real-time in response to different signals.