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Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange
Published on: April 27, 2017
Identifying Products of Recombinase-Mediated Cassette Exchange (RMCE) in Schizosaccharomyces pombe
Johanne M Murray1, Adam T Watson1, Antony M Carr1
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, E. Sussex BN1 9RQ, United Kingdom.
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.
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