Mice with Human SCN1A Alleles Generated by Recombinase-Mediated Cassette Exchange

描述（由申请人提供）：电压门控钠通道突变与几种不同临床严重程度的人类癫痫有关。SCN1A突变首次在全身性癫痫伴发热性癫痫发作（GEFS+）中被发现，这是一种良性的儿童期发病综合征，其家庭成员在儿童期有发热性癫痫发作，并可能在成年后发展为其他类型的癫痫发作。SCN1A突变也在婴儿严重肌阵挛性癫痫（SMEI）中被发现，SMEI是一种以全身性强直-阵挛或半阵挛发作为特征的婴儿发病综合征。随着综合征的发展，SMEI患者会出现其他类型的癫痫发作，包括肌阵挛性、缺失性和部分性癫痫发作，精神运动和智力发育下降。总的来说，在癫痫患者中报道了300多个SCN1A突变，使其成为癫痫最常见的遗传原因。SCN1A突变的体外表达研究揭示了多种功能缺陷。然而，异源表达系统中Nav1.1功能障碍与临床表型严重程度之间没有明显的相关性。缺乏明确的基因型-表型相关性可能反映了体外表达系统评估神经元钠通道突变的局限性。关于突变的功能后果的最可靠的数据可以从携带突变的转基因小鼠中获得。然而，同源重组产生敲入小鼠所需的资源和时间是令人望而却步的。重组介导的盒式交换（RMCE）允许快速有效地生产在靶位点携带突变dna的小鼠等位基因系列。在这种方法中，一个盒式受体包含一个可选择的标记，两侧有lox位点，通过同源重组靶向内源性小鼠位点。随后，通过在胚胎干细胞中介导的重组，将盒式受体交换为感兴趣的序列，这比同源重组要有效得多。效率的提高减少了产生多个变异所需的时间和资源，从而允许平行产生一系列小鼠的等位基因。特异性靶1将产生一种小鼠胚胎干细胞系，其中含有翻译起始位点的Scn1a外显子1通过同源重组被锁定盒受体取代。随后与SCN1A cDNA的交换将允许cDNA在内源性调控下表达，同时抑制小鼠基因的表达。特异性目标2将通过cre介导的盒式交换产生小鼠，其中小鼠Scn1a被人类Scn1a cDNA取代，作为该方法的关键原理验证实验。该方法的开发和验证将使人类癫痫突变的体内表征成为可能，并为理解癫痫的潜在机制提供宝贵的资源。

DESCRIPTION (provided by applicant): 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 Severe Myoclonic Epilepsy of Infancy (SMEI), an infant-onset syndrome characterized by generalized tonic-clonic or hemiclonic seizures. As the syndrome progresses, SMEI patients develop other seizure types including myoclonic, absence and partial seizures, and a decline of psychomotor and mental development. Overall more than 300 mutations of SCN1A have been reported in patients with epilepsy, making it the most common genetic cause of epilepsy. In vitro expression studies of SCN1A mutations have revealed a variety of functional defects. However, there is not an obvious correlation between Nav1.1 dysfunction in heterologous expression systems 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 knock-in mice by homologous recombination is prohibitive. 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. Specific aim 1 will generate a mouse ES cell line in which Scn1a exon 1 containing the translation start site is replaced by a loxed cassette acceptor via homologous recombination. Subsequent exchange with SCN1A cDNAs will allow expression of the cDNA under the endogenous regulatory control while ablating expression of the mouse gene. Specific aim 2 will generate mice by cre-mediated cassette exchange in which mouse Scn1a is replaced with the human SCN1A cDNA as a critical proof-of-principle experiment for this approach. Development and validation of this approach will enable in vivo characterization of human epilepsy mutations and provides a valuable resource for understanding the mechanisms underlying epilepsy. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to develop a system for rapid and efficient production of mouse models carrying human epilepsy mutations. Mouse models will provide insight into the molecular and genetic events that underlie epilepsy and will be valuable tools for developing novel therapeutic strategies.