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

描述（由申请人提供）：电压门控钠通道的突变与几种类型的人类癫痫有关，临床严重程度不同。SCN 1A的突变首先在全身性癫痫伴热性惊厥+（GEFS+）中发现，这是一种良性的儿童期发病综合征，其中家庭成员在儿童期有热性惊厥，并可能在成年后发展为其他癫痫发作类型。SCN 1A突变也已在婴儿严重肌阵挛性癫痫（SMEI）中发现，SMEI是一种婴儿发作的综合征，其特征为全身强直阵挛性或半阵挛性癫痫发作。随着综合征的进展，SMEI患者发展为其他癫痫发作类型，包括肌阵挛性、失神和部分性癫痫发作，以及精神和智力发育的下降。据报道，癫痫患者中有超过300种SCN 1A突变，使其成为癫痫最常见的遗传原因。SCN 1A突变的体外表达研究揭示了多种功能缺陷。然而，在异源表达系统中Nav1.1功能障碍与临床表型的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被工程化以携带突变的小鼠获得。然而，通过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。扩增介导的盒交换（RMCE）允许在靶基因座处携带突变DNA的小鼠的等位基因系列的快速且有效的产生。在该方法中，通过同源重组将含有侧翼为lox位点的选择性标记的盒受体靶向内源小鼠基因座。随后通过ES细胞中的cre介导的重组发生盒受体与目的序列的交换，这比同源重组有效得多。效率的提高减少了产生多个变体所需的时间和资源，允许平行产生小鼠的等位基因系列。特异性目的1将产生小鼠ES细胞系，其中含有翻译起始位点的Scn 1a外显子1通过同源重组被loxed盒受体取代。随后与SCN 1A cDNA的交换将允许在内源调节控制下表达cDNA，同时消除小鼠基因的表达。具体目标2将通过cre介导的盒交换产生小鼠，其中将小鼠Scn 1a替换为人SCN 1A cDNA，作为该方法的关键原理验证实验。这种方法的开发和验证将使人类癫痫突变的体内表征，并为了解癫痫的机制提供了宝贵的资源。 公共卫生相关性：该提案的目标是开发一种系统，用于快速有效地生产携带人类癫痫突变的小鼠模型。小鼠模型将提供深入了解癫痫的分子和遗传事件，并将是开发新的治疗策略的宝贵工具。

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.