In addition to building better hardware and software to image transcription in live cells, a crucial requirement of our single molecule experiments is a battery of bright, specific fluorescent reporters for each of the players in the process.
3D tracking individual molecules of Sox2 in the nucleus of a live mouse ES cell (30 ms per frame; bar, 5 um)(Chen, Zhang et al. 2014).
Over the years, our various labs have developed and/or optimized a host of labeling techniques to image individual components of the transcription process: chromatin modifications (Stasevich, Hayashi-Takanaka et al. 2014), Transcription Factors (Chen, Zhang et al. 2014), Components of the transcription machinery (Cisse, Izeddin et al. 2013), nascent mRNA (Lionnet, Czaplinski et al. 2011). We also collaborate with Luke Lavis to incorporate bright small fluorescent molecules in our assays (Grimm, English et al. 2015).
Live cell imaging of a primary mouse fibroblast expressing a labeled endogenous mRNA (Actb). Individual mRNA molecules appear as dim puncta that diffuse in the cytoplasm. Transcription of the Actb gene is induced with serum, resulting in accumulation of fluorescence at the two alleles (bright nuclear spots). (Lionnet, Czaplinski et al. 2011).
Using novel JF dyes synthesized in the Lavis lab to visualize the dynamics of a Transcription Factor (TetR single molecules, red) over the background of the nuclear chromatin (H2B superresolution map, green) (Grimm, English et al. 2015).
We continue to explore novel ways to design reporters constructs, lines and labels, optimizing many constraints: minimal genetic and functional disturbance, brightness, dynamic range, multiplexing ability, in order to eventually be able to monitor each step of the transcription process in live cells.