Generation of an Allelic Series of Knock-In Mice Using Recombinase-Mediated Cassette Exchange (RMCE)

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.

Cre Recombinase and Other Tyrosine Recombinases

“…For the production of therapeutic monoclonal antibodies, RMCE has streamlined the production of cell lines that allow rapid, efficient, and reliable expression of the antibody. , RMCE has also demonstrated to be useful to manipulate pest insects for research and field release and for exchanging large genomic regions in cells and in mice to replace a large endogenous genomic region precisely with the syntenic human sequence . Furthermore, RMCE was used to rapidly generate an allelic series of knock-in mice …”

“…234 Furthermore, RMCE was used to rapidly generate an allelic series of knock-in mice. 235 Another recent improvement of RMCE is the implementation into lentviral vectors for potential gene therapy applications 236,237 and the combination with other genomeediting technologies, such as TALENs 238 and CRISPR/Cas 239 (see also 5.8).…”

A RAGE Based Strategy for the Genome Engineering of the Human Respiratory Pathogen Mycoplasma pneumoniae

“…coli λ Red system, is needed to promote the cassette exchange, which limits its use in many bacterial species, including MPN. Cre recombinase, as well as other site-directed recombinases like Phi C31 or FLP, have been shown to be active in a number of bacterial species, but not many RMCE experiments have been performed in prokaryotes. − However, it is a common technology used for engineering the genome of animal cells. − In theory, this technology would be suited to any bacterial species for which the introduction of landing pads is possible. This also opens the possibility of creating and using YAC or BAC genomic DNA libraries of different bacteria to easily manipulate a desired fragment in yeast or in E.…”