Generation of a series of knock-in alleles using RMCE in ES cells

Recombinase-mediated cassette exchange (RMCE) is a powerful tool to generate a series of knock-in mutations into a particular gene of the mouse. It uses standard ES cell technologies to introduce an exchangeable cassette into the gene of interest by homologous recombination. Because the introduced exchangeable cassette is flanked by heterotypic specific recombination target sequences, site-specific recombinases can be used in a subsequent RMCE step to exchange the cassette by other sequences. Here, an experimental procedure for the application of RMCE in E14 ES cells using heterotypic FRT recombination target sequences and the site-specific recombinase Flp is elaborated.