Recombinase-mediated cassette exchange to rapidly and efficiently generate mice with human cardiac sodium channels.
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.
Liu
Kai
K
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0575, USA.
Hipkens
Susan
S
Yang
Tao
T
Abraham
Robert
R
Zhang
Wei
W
Chopra
Nagesh
N
Knollmann
Bjorn
B
Magnuson
Mark A
MA
Roden
Dan M
DM
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D018274:Electroporation; D053595:Embryonic Stem Cells; D040461:Gene Components; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D019426:Integrases; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015222:Sodium Channels / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; D015222:Sodium Channels; C045073:Cre recombinase; D019426:Integrases