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

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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.