Measuring transcription dynamics in living cells using a photobleaching approach

- Generating cell lines with tagged genes and mRNAs.
- Following transcription in living cells.
- Using a photobleaching approach to obtain transcription kinetics.

The transcriptional kinetics of RNA polymerase II, the enzyme responsible for mRNA transcription in the nucleoplasm, can be modulated by a variety of factors. It is therefore important to establish experimental systems that will enable the readout of transcription kinetics of specific genes as they occur in real time within individual cells. This can be performed by implementing fluorescent tagging of the mRNA under live-cell conditions. This chapter describes how to generate fluorescently tagged genes and mRNA, and how a photobleaching approach can produce information on mRNA transcription kinetics.