A High Throughput Imaging Assay for the Quantification of Gene Expression Dynamics at the Single Cell Level
Recorded On: 02/05/2018
The establishment and maintenance of gene expression programs is essential for cellular differentiation and organism development. For this reason, gene expression is tightly regulated at the level of mRNA transcription, splicing, and translation. Recently, a combination of genetically encoded fluorescent reporters capable of binding and visualizing mRNA transcripts in living cells, such as MS2 stem loops and MS2-GFP, and of image processing techniques to detect, track and measure these transcripts has enabled the characterization of the dynamic regulation of these processes in live cells. We will describe the design and implementation of a high-throughput imaging assay consisting of panels of cell lines stably expressing a variety of endogenous genes tagged with MS2-stem loops, automated live-cell confocal microscopy for the long-term visualization of the expression dynamics of these genes at the single allele level, automated image processing for cell and transcription site tracking in time-lapse series, and the generation of gene expression trajectories for hundreds of cells per sample. Furthermore, we will show practical implementations of this imaging-based assay to measure the transcriptional kinetics of several independently MS2-repeats-tagged genes, and to quantify changes in transcriptional on/off cycles for a glucocorticoid receptor (GR) regulated locus. Overall, the development of this approach opens the possibility of screening focused chemical or oligo siRNA libraries to identify and characterize novel molecular mechanisms regulating gene expression dynamics.
National Cancer Institute/NIH
Gianluca Pegoraro uses high-throughput imaging to identify and characterize molecular mechanisms regulating basic cellular processes such as nuclear architecture, gene expression and the DNA damage response.