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Methods Cell Biol. 2010;97:147-72. doi: 10.1016/S0091-679X(10)97009-X.

New and old reagents for fluorescent protein tagging of microtubules in fission yeast; experimental and critical evaluation.

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1
Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH93JR, United Kingdom.

Abstract

The green fluorescent protein (GFP) has become a mainstay of in vivo imaging in many experimental systems. In this chapter, we first discuss and evaluate reagents currently available to image GFP-labeled microtubules in the fission yeast Schizosaccharomyces pombe, with particular reference to time-lapse applications. We then describe recent progress in the development of robust monomeric and tandem dimer red fluorescent proteins (RFPs), including mCherry, TagRFP-T, mOrange2, mKate, and tdTomato, and we present data assessing their suitability as tags in S. pombe. As part of this analysis, we introduce new PCR tagging cassettes for several RFPs, new pDUAL-based plasmids for RFP-tagging, and new RFP-tubulin strains. These reagents should improve and extend the study of microtubules and microtubule-associated proteins in S. pombe.

PMID:
20719270
DOI:
10.1016/S0091-679X(10)97009-X
[Indexed for MEDLINE]

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