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

Lindhout, Beatrice I. ; Fransz, Paul ; Tessadori, Federico ; Meckel, Tobias ; Hooykaas, Paul J. J. ; Zaal, Engelbertus J. van der (2007)
Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins.
In: Nucleic acids research, 35 (16)
Article, Bibliographie

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. ; Fransz, Paul ; Tessadori, Federico ; Meckel, Tobias ; Hooykaas, Paul J. J. ; Zaal, Engelbertus J. van der
Type of entry: Bibliographie
Title: Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins
Language: English
Date: 2007
Journal or Publication Title: Nucleic acids research
Volume of the journal: 35
Issue Number: 16
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.

Divisions: 10 Department of Biology
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10 Department of Biology > Plant Membrane Biophyscis (20.12.23 renamed in Biology of Algae and Protozoa)
10 Department of Biology > Membrane Dynamics
Date Deposited: 06 Jun 2011 13:37
Last Modified: 23 Jul 2021 12:09
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