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

Lindhout, Beatrice I. and Fransz, Paul and Tessadori, Federico and Meckel, Tobias and Hooykaas, Paul J. J. and van der Zaal, Bert J. (2007):
Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins.
35, In: Nucleic acids research, (16), pp. e107, ISSN 1362-4962, [Article]

Abstract

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.

Item Type: Article
Erschienen: 2007
Creators: Lindhout, Beatrice I. and Fransz, Paul and Tessadori, Federico and Meckel, Tobias and Hooykaas, Paul J. J. and van der Zaal, Bert J.
Title: Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins.
Language: English
Abstract:

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.

Journal or Publication Title: Nucleic acids research
Volume: 35
Number: 16
Divisions: 10 Department of Biology
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10 Department of Biology > Plant Membrane Biophysics
10 Department of Biology > Membrane Dynamics
Date Deposited: 06 Jun 2011 13:37
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