Abstracts

Rationale: Mutations in voltage-gated sodium channels have been implicated in several types of human epilepsy with varying degrees of clinical severity. Mutations in SCN1A were first identified in Generalized Epilepsy with Febrile Seizures Plus (GEFS ), a benign, childhood-onset syndrome in which family members have febrile seizures in childhood and may go on to develop other seizure types as adults. SCN1A mutations have also been identified in Dravet Syndrome (Severe Myoclonic Epilepsy of Infancy), an infant-onset syndrome characterized by generalized tonic-clonic or hemiclonic seizures. As the syndrome progresses patients develop other seizure types including myoclonic, absence and partial seizures, and a decline of psychomotor and mental development. More than 700 mutations of SCN1A have been reported in patients with epilepsy, making it the most common genetic cause of epilepsy. Functional studies of SCN1A mutations in heterologous expression systems have revealed a variety of functional defects. However, there is not an obvious correlation between Nav1.1 dysfunction and severity of the clinical phenotype. The lack of a clear genotype-phenotype correlation may reflect a limitation of in vitro expression systems to evaluate neuronal sodium channel mutations. The most reliable data on functional consequences of mutations can be obtained from mice engineered to carry the mutations. However, the resources and time required for generating allelic series of knock-in mice by homologous recombination is prohibitive. Methods: Recombination-mediated cassette exchange (RMCE) allows for rapid and efficient production of an allele series of mice carrying mutant DNAs at the target locus. In this method, a cassette acceptor containing a selectable marker flanked by lox sites is targeted to the endogenous mouse locus by homologous recombination. Subsequent exchange of the cassette acceptor for the sequence of interest occurs by cre-mediated recombination in the ES cells, which is much more efficient than homologous recombination. The gain in efficiency decreases the time and resources required to generate multiple variants, allowing for parallel generation of an allelic series of mice. Results: We generated a mouse ES cell line in which Scn1a exon 1 containing the translation start site was replaced by a loxed cassette acceptor via homologous recombination. Subsequent exchange with SCN1A cDNAs allows expression of the human cDNA under the endogenous regulatory control while ablating expression of the mouse gene. Conclusions: This approach will enable in vivo characterization of human epilepsy mutations and provides a valuable resource for understanding the mechanisms underlying epilepsy and developing novel therapeutic strategies.