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Quantifying the transcriptional output of single alleles in single living mammalian cells

Abstract

Transcription kinetics of actively transcribing genes in vivo have generally been measured using tandem gene arrays. However, tandem arrays do not reflect the endogenous state of genome organization in which genes appear as single alleles. Here we present a robust technique for the quantification of mRNA synthesis from a single allele in real time in single living mammalian cells. The protocol describes how to generate cell clones harboring an MS2-tagged allele and how to detect in vivo transcription from this tagged allele at high spatial and temporal resolution throughout the cell cycle. Quantification of nascent mRNAs produced from the single tagged allele is performed using RNA fluorescence in situ hybridization (FISH) and live-cell imaging. Subsequent analyses and data modeling detailed in the protocol include measurements of transcription rates of RNA polymerase II, determination of the number of polymerases recruited to the tagged allele and measurement of the spacing between polymerases. Generation of the cells containing the single tagged alleles should take up to 1 month; RNA FISH or live-cell imaging will require an additional week.

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Figure 1: Generation of a cell system expressing a single GOI-MS2 allele.
Figure 2: Detection and quantification of GOI-MS2 mRNAs by RNA FISH.
Figure 3: Following transcription kinetics of the GOI-MS2 allele in real-time.

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Acknowledgements

This work was supported by grants from the European Research Council (ERC), the Israel Cancer Research Fund (ICRF), and the Union for International Cancer Control (UICC) to Y.S.-T., and an Israel Science Foundation (ISF) Bikura grant to Y.G. and Y.S.-T.

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Authors

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S.Y., L.R., Y.G. and Y.S.-T. wrote the paper.

Corresponding author

Correspondence to Yaron Shav-Tal.

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Supplementary information

Supplementary Sequence Archive

Full sequence for pSL24XMS2 plasmid (TXT 5 kb)

Supplementary Video 1

Fluctuations in transcriptional activity observed in a living cell. A time-lapse movie of a cell expressing the MS2-GFP protein showing the fluctuations in activity of the transcribing allele over a period of 196 minutes (imaged every 4 minutes). (AVI 82 kb)

Supplementary Video 2

Recovery of mRNA signal in a FRAP experiment. A FRAP movie of a cell expressing the MS2-GFP protein showing the recovery in fluorescence of the transcribing allele after photobleaching (imaged every 6 minutes after photobleaching). (AVI 181 kb)

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Yunger, S., Rosenfeld, L., Garini, Y. et al. Quantifying the transcriptional output of single alleles in single living mammalian cells. Nat Protoc 8, 393–408 (2013). https://doi.org/10.1038/nprot.2013.008

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