Recombinase‐mediated cassette exchange to rapidly and efficiently generate mice with human cardiac sodium channels

Abstract

SCN5A encodes the predominant voltage-gated sodium channel isoform in human heart and nearly 100 variants have now been described and studied in vitro. However, development of animal models to analyze function of such large numbers of human gene variants represents a continuing challenge in translational medicine. Here, we describe the implementation of a two stage procedure, recombinase-mediated cassette exchange (RMCE), to efficiently and rapidly generate mice in which a full-length human cDNA replaces expression of the murine ortholog. In the first step of RMCE, conventional homologous recombination in mouse ES cells was used to replace scn5a exon 2 (that contains the translation start site) with a cassette acceptor that includes the thymidine kinase gene, flanked by loxP/inverted loxP sites. In the second step, the cassette acceptor site was replaced by the full-length wild-type human SCN5A cDNA by Cre/loxP-mediated recombination. The exchange event occurred in 7/29 (24%) colonies, and the time from electroporation to first homozygotes was only 8 months. PCR-restriction fragment length polymorphism (RFLP) showed that the murine isoform was replaced by the human one, and functional studies indicated that mice with human cardiac sodium channels have wild-type sodium current density, action potential durations, heart rates, and QRS durations. These data demonstrate that RMCE can be used to generate mice in which a targeted allele can be rapidly and efficiently replaced by variants of choice, and thereby can serve as an enabling approach for the functional characterization of ion channel and other DNA variants.

Recombinase-mediated cassette exchange reveals the selective use of Gq/G11-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development

“…Indeed, the efficiency of RMCE-mediated knock-in recombination was very high in our screening (44 to 87%), compared with conventional homologous recombination. This is similar to or higher than the efficiencies previously reported for RMCE using the Cre-lox system (Araki et al, 2006;Liu et al, 2006;Toledo et al, 2006) or the Flp-FRT system (Cesari et al, 2004;Roebroek et al, 2006). In our RMCE procedure, we replaced a 1.0 kb genomic sequence with knock-in sequences containing mutant lox sites and an FRT-flanked Puro.…”

Striking In Vivo Phenotype of a Disease-Associated Human SCN5A Mutation Producing Minimal Changes in Vitro

“…21 The data demonstrate that D1275N causes a severe defect in sodium channel function in vivo , consistent with the reported clinical phenotypes.…”

“…We have previously modified the Scn5a locus in mouse embryonic stem cells to enable the technique of recombinase mediated cassette exchange. 21–23 In our initial studies, we inserted the full-length human SCN5A cDNA into the targeted locus. 21 Mice homozygous for the exchanged allele (termed H/H) expressed only the human allele and had normal electrocardiograms (ECGs) and ventricular sodium current, supporting the hypothesis that expression of the exchanged allele was under control of endogenous Scn5a regulatory mechanisms.…”

“…To address this discrepancy, we have used recombinase-mediated cassette exchange 21 to engineer mice expressing the mutant human channel (here termed DN); we compared the functional properties of these animals to those expressing wild-type human alleles (H), that we previously generated in an identical fashion. 21 The data demonstrate that D1275N causes a severe defect in sodium channel function in vivo , consistent with the reported clinical phenotypes.…”

Bio-applications Derived from Site-Directed Genome Modification Technologies

“…This technology, referred to as 'recombinase-mediated cassette exchange' (RMCE), includes loxP sites [Cre recombinase (Liu et al 2006a ;Sandhu et al 2011 ) ] or FRT sites [Flp recombinase (Turan et al 2010 ;Roebroek et al 2011 ) , as in the Flp-in TM system developed by Invitrogen, http://products.invitrogen.com/ivgn/product/K601001 ]. To be effective, these recombinases must be used at very high concentrations, which, in the case of Cre, triggers endonucleolytic activity and therefore cellular toxicity.…”