[Protocol] Identifying products of recombinase-mediated cassette exchange (RMCE) in schizosaccharomyces pombe

Murray, Johanne, Watson, Adam and Carr, Antony (2016) [Protocol] Identifying products of recombinase-mediated cassette exchange (RMCE) in schizosaccharomyces pombe. Cold Spring Harbor Protocols, 2016 (5). ISSN 1559-6095

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Abstract

Homologous recombination is highly efficient when mediated between two identical target sequences by recombination enzymes such as Cre. Exploiting this, recombinase-mediated cassette exchange (RMCE) was developed for the genetic manipulation of eukaryotic cells, including those of Schizosaccharomyces pombe. RMCE can be summarized in three stages: (1) A loxP-ura4+-loxM3 cassette is introduced into the genome using standard homologous recombination techniques to create a “base strain.” (2) A Cre-expression plasmid carrying a protein tag or replacement gene flanked by loxP and loxM3 is introduced into the cell. (3) Cassette exchange between the chromosomal cassette and the plasmid cassette results in either gene tagging or gene replacement. This is selected for by loss of the marker. This protocol explains how to identify the products of the exchange events in the last stage.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Research Centres and Groups: Genome Damage and Stability Centre
Subjects: Q Science > Q Science (General)
Depositing User: Sarah Frances
Date Deposited: 12 Dec 2017 13:54
Last Modified: 26 Mar 2018 13:34
URI: http://sro.sussex.ac.uk/id/eprint/71863

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