Skip Navigation



Nucleic Acids Research Advance Access published online on August 17, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm618
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (6900K) Freely available
Right arrow Screen PDF (607K) Freely available
Right arrowOA All Versions of this Article:
35/16/e107    most recent
gkm618v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Lindhout, B. I.
Right arrow Articles by Zaal, B. J. v. d.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lindhout, B. I.
Right arrow Articles by Zaal, B. J. v. d.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins

Beatrice I. Lindhout1, Paul Fransz2, Federico Tessadori2, Tobias Meckel3, Paul J.J. Hooykaas1 and Bert J. van der Zaal1,*

1Institute of Biology Leiden, Department of Molecular and Developmental Genetics, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, 2Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM, Amsterdam and 3Physics of Life Processes, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA, Leiden, The Netherlands

*To whom correspondence should be addressed. Tel: +31 71 527 4760; Fax: +31 71 527 4999; Email: b.j.v.d.zaal{at}biology.leidenuniv.nl

Received June 13, 2007. Accepted July 28, 2007.

Several techniques are available to study chromosomes or chromosomal domains in nuclei of chemically fixed or living cells. Current methods to detect DNA sequences in vivo are limited to trans interactions between a DNA sequence and a transcription factor from natural systems. Here, we expand live cell imaging tools using a novel approach based on zinc finger-DNA recognition codes. We constructed several polydactyl zinc finger (PZF) DNA-binding domains aimed to recognize specific DNA sequences in Arabidopsis and mouse and fused these with GFP. Plants and mouse cells expressing PZF:GFP proteins were subsequently analyzed by confocal microscopy. For Arabidopsis, we designed a PZF:GFP protein aimed to specifically recognize a 9-bp sequence within centromeric 180-bp repeat and monitored centromeres in living roots. Similarly, in mouse cells a PZF:GFP protein was targeted to a 9-bp sequence in the major satellite repeat. Both PZF:GFP proteins localized in chromocenters which represent heterochromatin domains containing centromere and other tandem repeats. The number of PZF:GFP molecules per centromere in Arabidopsis, quantified with near single-molecule precision, approximated the number of expected binding sites. Our data demonstrate that live cell imaging of specific DNA sequences can be achieved with artificial zinc finger proteins in different organisms.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.