Linked Life Data

17083109

Source:http://linkedlifedata.com/resource/pubmed/id/17083109

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
2006-11-8
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1526-954X
pubmed:author
pubmed:issnType
Print
pubmed:volume
44
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
556-64
pubmed:dateRevised
2011-7-22
pubmed:meshHeading
pubmed-meshheading:17083109-Animals, pubmed-meshheading:17083109-DNA, Complementary, pubmed-meshheading:17083109-DNA Primers, pubmed-meshheading:17083109-Electroporation, pubmed-meshheading:17083109-Embryonic Stem Cells, pubmed-meshheading:17083109-Gene Components, pubmed-meshheading:17083109-Gene Transfer Techniques, pubmed-meshheading:17083109-Genetic Vectors, pubmed-meshheading:17083109-Humans, pubmed-meshheading:17083109-Integrases, pubmed-meshheading:17083109-Mice, pubmed-meshheading:17083109-Mice, Transgenic, pubmed-meshheading:17083109-Models, Animal, pubmed-meshheading:17083109-Muscle Proteins, pubmed-meshheading:17083109-Polymorphism, Restriction Fragment Length, pubmed-meshheading:17083109-Reverse Transcriptase Polymerase Chain Reaction, pubmed-meshheading:17083109-Sodium Channels
pubmed:year
2006
pubmed:articleTitle
Recombinase-mediated cassette exchange to rapidly and efficiently generate mice with human cardiac sodium channels.
pubmed:affiliation
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0575, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural