Generation of Scn1a-Targeted Rat Model

A pair of TALENs targeting exon 1 of rat Scn1a (Ensembl: ENSRNOG00000053122) was designed and constructed using a two-step assembly method with a Platinum Gate kit as previously reported (22 (link)). Assembled sequence was 5′-TGCAGGATGACAAGATGgagcaaacagtgcttGTACCACCAGGACCTGA-3′, where uppercase and lowercase letters indicate TALEN target sequences and spacer sequence, respectively. TALENs were microinjected into fertilized eggs of Fisher 344 (F344) rats and transferred into the oviducts of pseudopregnant female Wistar rats, as previously described (23 (link)). Genomic DNA was extracted from the tail using the GENEXTRACTOR TA-100 automatic DNA purification system (Takara Bio) and amplified with specific primer sets (forward 5′-TCCTCACTTGTTGGGTCTCA-3′, reverse 5′-TCAGGGTGACTTCAGCATTTC-3′). The polymerase chain reaction (PCR) products were directly sequenced using the BigDye terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Life Technologies). Rats from the fifth generation or later were used in all experiments. Genotypes were assessed by running the PCR products from ear DNA on a Caliper electrophoresis system at postnatal day (P9). Only male rats were used in experiments to eliminate sex differences.