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

Sharon Yunger, Liat Rosenfeld, Yuval Garini, Yaron Shav-Tal

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

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.

Original languageEnglish
Pages (from-to)393-408
Number of pages16
JournalNature Protocols
Volume8
Issue number2
DOIs
StatePublished - Feb 2013

Bibliographical note

Funding Information:
acknowleDGMents 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.

Funding

acknowleDGMents 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.

FundersFunder number
Israel Cancer Research Fund
International Union Against Cancer
Union for International Cancer Control
European Research Council
Israel Science Foundation

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