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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 CarrHomologous 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 ProtocolsISSN
1559-6095Publisher
Cold Spring Harbor Laboratory PressExternal DOI
Issue
5Volume
2016Department 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
2017-12-12First Compliant Deposit (FCD) Date
2017-12-12Usage metrics
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