title: Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins.
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.
authors: Institute of Biology Leiden, Department of Molecular and Developmental Genetics, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.
authors: Beatrice I
authors: BI
authors: Lindhout
authors: Paul
authors: P
authors: Fransz
authors: Federico
authors: F
authors: Tessadori
authors: Tobias
authors: T
authors: Meckel
authors: Paul J J
authors: PJ
authors: Hooykaas
authors: Bert J
authors: BJ
authors: van der Zaal
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D002503:Centromere / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D049408:Luminescent Agents / Q000032:analysis*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D018517:Plant Roots / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D016335:Zinc Fingers
chemical_list: D049408:Luminescent Agents; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA
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