Generation of <i>Scn9a</i>-N641Y knockin mice.

<p>Schematic representation of the (A) wild-type allele, (B) targeting construct introduced into embryonic stem (ES) cells. Numbered boxes denote exons; *, p.N641Y missense change introduced into exon 11; PCRa and PCRb, primers used to screen ES cell DNA for homologous recombination; S and probe, denotes SspI sites and probe used in genomic Southern blot of ES cells; ACN cassette, Cre-recombinase gene (<i>Cre</i>) driven by the testes-specific promoter from the angiotensin-converting enzyme gene (<i>tACE</i>); <i>Cre</i> is linked to the <i>Neo</i>^r selectable marker driven by the mouse RNA polymerase II large subunit gene (<i>polII</i>); the entire cassette is flanked by 34 bp loxP sites oriented in parallel. TK, HSV-TK gene for negative selection of ES cells. (C) following <i>Cre</i>-mediated self-excision in the chimeric mouse germline, a single loxP site and the point mutation remain. (D) Southern blot of three SspI cut ES cell clones followed by hybridization of probe yields an 8.4 kb endogenous band and a 7.2 kb targeted band (horizontal arrows); vertical arrow denotes clone used to make mouse. (E) LightScanner normalized melting peaks used to genotype <i>SCN9A</i>^+/+, <i>SCN9A</i>^N641Y/+, and <i>SCN9A</i>^N641Y/N641Y mice. (F) PCR used to verify self-excision of the ACN cassette. Amplicons generated by primers flanking remaining 34 bp loxP site in intron 10 yield distinct <i>SCN9A</i>^+/+ (left), <i>SCN9A</i>^N641Y/+ (center), and <i>SCN9A</i>^N641Y/N641Y (right, denoted by arrow) bands on 2% agarose.