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Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol   

Edited by
Yanjie Li
Reviewed by
Anonymous reviewer
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Original research article

A brief version of this protocol appeared in:
Yeast
Dec 2015

Abstract

The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.

Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, Schizosaccharomyces pombe. In the following we describe how to swap the ura4+ marker to a kanMX6, natMX4, or hphMX4 marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the MX markers for nutritional markers, such as arg3+, his3+, leu1+ and ura4+.

Keywords: Schizosaccharomyces pombe, Selectable marker, Marker switch, Li-Acetate transformation, Gene tagging, Gene deletion, Genetic manipulation

Background

This single-step marker swap protocol for Schizosaccharomyces pombe allows for any tagged or mutated gene marked with an MX-type antibiotic marker to be swapped for a nutritional marker (cassettes containing the arg3+, his3+, leu1+, and ura4+ have been constructed) and to exchange genetic ura4+-markers for any MX-type antibiotic resistance marker (kanMX, natMX, and hphMX constructs have been tested for this study) (Lorenz et al., 2015a). Previously, this kind of approach was only feasible for MX-type antibiotic resistance markers (Sato et al., 2005; Hentges et al., 2005). Exchanging antibiotic resistance markers for each other already represented a basic set of useful genetic tools, the ura4+-to-MX as well as the arg3MX4, his3MX4, leu1MX4, and ura4MX4 marker swap cassettes expand this genetic toolbox for tagging and mutating genes in fission yeast (Lorenz et al., 2015a). The lithium acetate transformation protocol itself was described previously (Keeney and Boeke, 1994) and recently suggested modifications (http://listserver.ebi.ac.uk/pipermail/pombelist/2014/004012.html) were incorporated to provide a highly efficient procedure. Streamlining Schizosaccharomyces pombe strain construction in this way is time-saving and, therefore, will prove useful for fission yeast researchers.

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Brown, S. D. and Lorenz, A. (2016). Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol. Bio-protocol 6(24): e2075. DOI: 10.21769/BioProtoc.2075.
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