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Time-resolved analysis of transcription kinetics in single live mammalian cells
Cited 1 time in
Web of Science
Cited 1 time in Scopus
- Authors
- Issue Date
- 2022-11
- Publisher
- Frontiers Media S.A.
- Citation
- Frontiers in Physics, Vol.10, p. 977125
- Abstract
- In eukaryotic cells, RNA polymerase II synthesizes mRNA in three stages, initiation, elongation, and termination, and numerous factors determine how quickly a gene is transcribed to produce mRNA molecules through these steps. However, there are few techniques available to measure the rate of each step in living cells, which prevents a better understanding of transcriptional regulation. Here, we present a quantitative analysis method to extract kinetic rates of transcription from time-lapse imaging data of fluorescently labeled mRNA in live cells. Using embryonic fibroblasts cultured from two knock-in mouse models, we monitored transcription of beta-actin and Arc mRNA labeled with MS2 and PP7 stem-loop systems, respectively. After inhibiting transcription initiation, we measured the elongation rate and the termination time by fitting the time trace of transcription intensity with a mathematical model function. We validated our results by comparing them with those from an autocorrelation analysis and stochastic simulations. This live-cell transcription analysis method will be useful for studying the regulation of elongation and termination steps, providing insight into the diverse mechanisms of transcriptional processes.
- ISSN
- 2296-424X
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