The spatial organization of the genome within the nucleus is now seen as a key contributor to genome function. Studying chromatin dynamics in living cells has been rendered possible by the development of fast microscopy coupled with fluorescent repressor operator systems (FROS). In these systems, arrays of protein-binding sites integrated at specific loci by homologous recombination are monitored through the fluorescence of tagged DNA-binding proteins. In the budding yeast, where homologous recombination is efficient, this technique, combined with targeting assay and genetic analysis, has been extremely powerful for studying the determinants and function of chromatin dynamics in living cells. However, issues have been recurrently raised in different species regarding the use of these systems. Here we discuss the different uses of gene tagging with FROS and their limitations, focusing in budding yeast as a model organism.
Keywords: FROS; Saccharomyses cerevisiae; lacO; nuclear organization; tetO.
Copyright © 2014 John Wiley & Sons, Inc.