Zinc finger-mediated live cell imaging in Arabidopsis roots.
Following the elucidation of recognition codes, artificial zinc finger (ZF) domains can now be assembled to create custom-made DNA-binding proteins in which the alpha helix of each zinc finger mediates an interaction with 3 or 4 bp of DNA. A module of consecutive zinc finger domains, designated a polydactyl zinc finger (PZF) domain, is thus capable of binding an extended number of base pairs of DNA. Besides the multitude of utilities of PZF domains addressed in other chapters, we have shown that they can also be used for live cell imaging of repetitive DNA sequences in Arabidopsis, as well as in mouse cells by generating and expressing PZF:GFP fusion proteins (1). Here we provide a detailed protocol for the construction of such PZF:GFP reporter proteins using our established cloning vehicles, together with a protocol for their expression in plants in order to achieve in vivo labelling of repetitive DNA. Furthermore we provide an accurate quantification method for GFP signals using fluorescent beads (FluoSpheres). Single-molecule precision can be obtained using any confocal setup once the fluorescent beads have been calibrated against purified GFP. The methods can easily be adapted to meet the demands for other situations or for other experimental systems.
Lindhout
Beatrice I
BI
Clusius Laboratory, Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, Leiden, The Netherlands.
Meckel
Tobias
T
van der Zaal
Bert J
BJ
D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D016335:Zinc Fingers / Q000235:genetics*
D049452:Green Fluorescent Proteins