Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Genetics & Heredity, Knock-in, Mutation, Recombinase-mediated cassette exchange, RMCE, Site-specific recombinase, FRT, Flp, LoxP, Cre, FLP-RECOMBINASE, ES CELLS, LRP1, MOTIF, LOCI, Alleles, Animals, Cell Line, DNA Nucleotidyltransferases, Embryonic Stem Cells, Gene Knock-In Techniques, Integrases, Mice, Mutagenesis, Insertional, Plasmids, Recombinases, 0399 Other Chemical Sciences, 0601 Biochemistry and Cell Biology, Developmental Biology, 3101 Biochemistry and cell biology, 3404 Medicinal and biomolecular chemistry

Abstract:

Molecular genetic strategies applying embryonic stem cell (ES cell) technologies to study the function of a gene in mice or to generate a mouse model for a human disease are continuously under development. Next to (conditional) inactivation of genes the application and importance of approaches to generate knock-in mutations are increasing. In this chapter the principle and application of recombinase-mediated cassette exchange (RMCE) are discussed as being a new emerging knock-in strategy, which enables easy generation of a series of different knock-in mutations within one gene. An RMCE protocol, which was used to generate a series of different knock-in mutations in the Lrp1 gene of ES cells, is described in detail as an example of how RMCE can be used to generate highly efficiently an allelic series of differently modified ES cell clones from a parental modified ES cell clone. Subsequently the differently modified ES cell clones can be used to generate an allelic series of mutant knock-in mice.