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

journal contribution
posted on 2023-06-09, 09:13 authored by Jo Murray, Adam WatsonAdam Watson, Antony CarrAntony Carr
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.

Funding

Smc5/6 and replication fork stability; G0179; MRC-MEDICAL RESEARCH COUNCIL; G0901011

Replication stalling in a palindrome; G0181; ASSOCIATION FOR INTERNATIONAL CANCER RESEARCH; 10-0273

History

Publication status

  • Published

File Version

  • Accepted version

Journal

Cold Spring Harbor Protocols

ISSN

1559-6095

Publisher

Cold Spring Harbor Laboratory Press

Issue

5

Volume

2016

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Research groups affiliated with

  • Genome Damage and Stability Centre Publications

Full text available

  • No

Peer reviewed?

  • No

Legacy Posted Date

2017-12-12

First Compliant Deposit (FCD) Date

2017-12-12

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