Advertisement

Visualizing Nucleic Acids in Living Cells by Fluorescence In Vivo Hybridization

  • Joop Wiegant
  • Anneke K. Brouwer
  • Hans J. Tanke
  • Roeland W. DirksEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 659)

Abstract

The analysis of the spatial–dynamic properties of DNA and RNA molecules in living cells will greatly extend our knowledge of genome organization and gene expression regulation in the cell nucleus. The development of hybridization methods allowing detection of specific endogenous DNA and RNA sequences in living cells has therefore been a challenge for many years. However, there are many technical issues that have proven so far to be difficult, or even impossible, to overcome. As a result, in most situations, the application of in vivo hybridization methods is currently limited to the visualization of highly repetitive DNA sequences or abundant RNA species. We describe a protocol that enables the visualization and tracking of telomeres in living cells by hybridization with a fluorescent peptide nucleic acid (PNA) probe. Furthermore, we describe a method that allows the detection of abundant endogenous RNAs in living cells by microinjecting fluorescently labeled complementary 2′-O-methyl RNA probes.

Key words

Microinjection Glass bead loading In vivo hybridization Peptide nucleic acid 2′-O-Me RNA Live cell imaging 

Notes

Acknowledgments

We wish to thank Dr. K. Vang Nielsen for providing PNA probes. This work was supported by Cyttron grant no. BSIK03036.

References

  1. 1.
    Lanctôt, C., Cheutin, T., Cremer, M., Cavalli, G., and Cremer, T. (2007) Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nature Rev Genet 8, 104–115.PubMedCrossRefGoogle Scholar
  2. 2.
    Foster, H. A., and Bridger, J. M. (2005) The genome and the nucleus: a marriage made by evolution. Genome organisation and nuclear architecture. Chromosoma 114, 212–229.PubMedCrossRefGoogle Scholar
  3. 3.
    Bolzer, A., Kreth, G., Solovei, I., Koehler, D., Saracoglu, K., Fauth, C., et al. (2005) Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PloS Biol 3, e157.PubMedCrossRefGoogle Scholar
  4. 4.
    Meaburn, K.J., and Misteli, T. (2008) Locus-specific and activity-independent gene repositioning during early tumorigenesis. J Cell Biol 180, 39–50.PubMedCrossRefGoogle Scholar
  5. 5.
    Dirks, R.W., Hattinger, C.M., Molenaar, C., and Snaar, S.P. (1999) Synthesis, processing, and transport of RNA within the three-dimensional context of the cell nucleus. Crit Rev Eukaryot Gene Expr 9, 191–201.PubMedCrossRefGoogle Scholar
  6. 6.
    Smith, K.P., Byron, M., Johnson, C., Xing, Y., and Lawrence, J.B. (2007) Defining early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains. J Cell Biol 178, 951–964.PubMedCrossRefGoogle Scholar
  7. 7.
    Trinkle-Mulcahy, L., and Lamond, A.I. (2008) Nuclear functions in space and time: Gene expression in a dynamic, constrained environment. FEBS Lett 582, 1960–1970.PubMedCrossRefGoogle Scholar
  8. 8.
    Dirks, R.W., and Tanke, H.J. (2006) Advances in fluorescent tracking of nucleic acids in living cells. Biotechniques 40, 489–496.PubMedCrossRefGoogle Scholar
  9. 9.
    Santangelo, P., Nitin, N., and Bao, G. (2006) Nanostructured probes for RNA detection in living cells. Ann Biomed Eng 34, 39–50.PubMedCrossRefGoogle Scholar
  10. 10.
    Dirks, R.W., Molenaar, C., and Tanke, H.J. (2003) Visualizing RNA molecules inside the nucleus of living cells. Methods 29, 51–57.PubMedCrossRefGoogle Scholar
  11. 11.
    McNeil, P.L., and Warder, E. (1987) Glass beads load macromolecules into living cells. J Cell Sci 88, 669–678.PubMedGoogle Scholar
  12. 12.
    Molenaar, C., Marras, S.A., Slats, J.C.M., Truffert, J.-C., Lemaître, M., Raap, A.K., Dirks, R.W., and Tanke, H.J. (2001) Linear 2′ O-Methyl RNA probes for the visualization of RNA in living cells. Nucleic Acids Res 29, e89.PubMedCrossRefGoogle Scholar
  13. 13.
    Molenaar, C., Wiesmeijer, K., Verwoerd, N.P., Khazen, S., Eils, R., Tanke, H.J., and Dirks, R.W. (2003) Visualizing telomere dynamics in living mammalian cells using PNA probes. EMBO J 22, 6631–6641.PubMedCrossRefGoogle Scholar
  14. 14.
    Molenaar, C., Abdulle, A., Gena, A., Tanke, H.J., and Dirks, R.W. (2004) Poly(A)+ RNAs roam the cell nucleus and pas through speckle domains in transcriptionally active and inactive cells. J Cell Biol 165, 191–202.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Joop Wiegant
    • 1
  • Anneke K. Brouwer
    • 1
  • Hans J. Tanke
    • 1
  • Roeland W. Dirks
    • 1
    Email author
  1. 1.Department of Molecular Cell BiologyLeiden University Medical CentreLeidenThe Netherlands

Personalised recommendations