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Development of rat models for human epilepsy by methods of gene modification

通过基因修饰方法开发人类癫痫大鼠模型

基本信息

批准号：
16200029
负责人：
SERIKWA Tadao
金额：
$ 30.62万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16200029/
关键词：
Rat Epilepsy Mutagenesis Etyl nitrosourea Disease model Chlorambucil Epilepsy gene Gene-modified rat

项目摘要

The aim of this study was the generation and partial characterization of mutant rats that show alteration in epilepsy related genes by screening of ENU- or chlorambucil-induced mutants. Although chlorambucil turned out to be not very efficient in our approach, a recessive mutation with a dwarf phenotype was obtained and is maintained by full-sib mating. We have furthermore developed a new, efficient approach that combines two methods : a high-throughput, low-cost screening assay that uses the phage Mu transposition reaction, and intracytoplasmic sperm injection (ICSI) for the recovery of the rare heterozygous genotypes from our newly generated frozen sperm repository, the Kyoto University Rat Mutant Archive (KURMA). This Mu transposition reaction can be combined with DNA pooling and therefore facilitates an efficient screening approach for mutagenized animals, termed MuT-POWER (Mu Transpositon Poling method With sequencer). The number of G1 DNA and sperm samples has already expanded to 5,000, and we could find 18 point mutations in epilepsy-related genes. Rats with a missense mutation in sodium channel SONIA gene were recovered from cryopreserved sperm by ICSI and a strain with the mutation has been generated. The rats showed a high sensitivity for pentylenetetrazol-induced convulsions and hot-water-induced convulsion, suggesting that they would be possible models for human heat-sensitive convulsions or GEFS+ patients. KURMA virtually allows the production of any rat model resembling human diseases.

本研究的目的是通过筛选ENU或苯丁酸氮芥诱导的突变体，产生和部分表征癫痫相关基因发生改变的突变大鼠。虽然苯丁酸氮芥在我们的方法中不是很有效，但获得了一个具有侏儒表型的隐性突变，并通过全同胞交配来维持。我们还开发了一种新的，有效的方法，结合了两种方法：一个高通量，低成本的筛选试验，使用噬菌体Mu转座反应，和胞浆内精子注射（ICSI）的罕见的杂合基因型的回收从我们新产生的冷冻精子库，京都大学大鼠突变档案（KURMA）。这种Mu转座反应可以与DNA合并相结合，因此有助于诱变动物的有效筛选方法，称为MuT-POWER（带有测序仪的Mu转座极化方法）。G1期DNA和精子样本的数量已经扩大到5,000个，我们可以在癫痫相关基因中发现18个点突变。通过ICSI从冷冻保存的精子中回收钠通道SONIA基因错义突变的大鼠，并产生了具有突变的品系。大鼠对戊四氮诱导的惊厥和热水诱导的惊厥表现出高敏感性，这表明它们可能是人类热敏感性惊厥或GEFS+患者的模型。KURMA实际上允许生产任何类似人类疾病的大鼠模型。