Advertisement

Preparation of Cell Lines for Single-Cell Analysis of Transcriptional Activation Dynamics

  • Ilona U. Rafalska-Metcalf
  • Susan M. Janicki
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 977)

Abstract

Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

Key words

Live-cell imaging Transgene array Stable-cell lines Transcription Chromatin 

References

  1. 1.
    Rafalska-Metcalf IU, Janicki SM (2007) Show and tell: visualizing gene expression in living cells. J Cell Sci 120:2301–2307PubMedCrossRefGoogle Scholar
  2. 2.
    Wu B, Piatkevich KD, Lionnet T, Singer RH, Verkhusha VV (2011) Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics. Curr Opin Cell Biol 23:310–317PubMedCrossRefGoogle Scholar
  3. 3.
    Darzacq X, Yao J, Larson DR, Causse SZ, Bosanac L, de Turris V, Ruda VM, Lionnet T, Zenklusen D, Guglielmi B, Tjian R, Singer RH (2009) Imaging transcription in living cells. Annu Rev Biophys 38:173–196PubMedCrossRefGoogle Scholar
  4. 4.
    Belmont AS (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11:250–257PubMedCrossRefGoogle Scholar
  5. 5.
    Keryer-Bibens C, Barreau C, Osborne HB (2008) Tethering of proteins to RNAs by bacteriophage proteins. Biol Cell 100:125–138PubMedCrossRefGoogle Scholar
  6. 6.
    Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333:642–646PubMedCrossRefGoogle Scholar
  7. 7.
    Tsukamoto T, Hashiguchi N, Janicki SM, Tumbar T, Belmont AS, Spector DL (2000) Visualization of gene activity in living cells. Nat Cell Biol 2:871–878PubMedCrossRefGoogle Scholar
  8. 8.
    Janicki SM, Tsukamoto T, Salghetti SE, Tansey WP, Sachidanandam R, Prasanth KV, Ried T, Shav-Tal Y, Bertrand E, Singer RH, Spector DL (2004) From silencing to gene expression: real-time analysis in single cells. Cell 116:683–698PubMedCrossRefGoogle Scholar
  9. 9.
    Rafalska-Metcalf IU, Powers SL, Joo LM, LeRoy G, Janicki SM (2010) Single cell analysis of transcriptional activation dynamics. PLoS One 5:e10272PubMedCrossRefGoogle Scholar
  10. 10.
    Masui O, Bonnet I, Le Baccon P, Brito I, Pollex T, Murphy N, Hupe P, Barillot E, Belmont AS, Heard E (2011) Live-cell chromosome dynamics and outcome of X chromosome pairing events during ES cell differentiation. Cell 145:447–458PubMedCrossRefGoogle Scholar
  11. 11.
    Shav-Tal Y, Darzacq X, Shenoy SM, Fusco D, Janicki SM, Spector DL, Singer RH (2004) Dynamics of single mRNPs in nuclei of living cells. Science 304:1797–1800PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Ilona U. Rafalska-Metcalf
    • 1
  • Susan M. Janicki
    • 1
  1. 1.Molecular and Cellular Oncogenesis ProgramThe Wistar InstitutePhiladelphiaUSA

Personalised recommendations