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Fig. 1. Generation of an allelic series of Sox2 mutations by gene targeting. (A) A schematic diagram of the Sox2 locus (top) and targeting vector used to generate the Sox2^ENHneo regulatory mutant allele (middle) in ES cells. From this mutant allele, Sox2^ENHneo is obtained by in vivo Cre-mediated excision of the neo cassette. The Sox2^ß-geo null/reporter allele (bottom) was previously described (Zappone et al., 2000). The PGKneo and PGKTK cassettes are derived from the pPNT vector. ENH is the DNA region containing the brain enhancer described by Zappone et al. (Zappone et al., 2000); ,ß and are probes used for Southern analysis [for details of and see Zappone et al. (Zappone et al., 2000)], ß is an AccI-XbaI 750 nucleotide fragment within the Sox2 coding region; E, EcoRI; S, SalI; N, NotI; H3, HindIII; X, XhoI; Xb, XbaI. Grey triangles indicate loxP sites; arrows indicate PCR primers used for verifying PGKneo deletion. (B,C) Southern analysis of EcoRI (B) and XbaI (C) digests of ES cell clones. In B, probe detects a 16 kb band from the wild-type allele, a 12.5 kb band from the Sox2^ENHneo allele, and a 6.5 kb band from the Sox2^ß-geo allele. In C, probe ß detects a 14 kb band from the wild-type allele, and a 7.2 kb band from the Sox2^ENHneo allele. Cre-mediated in vivo deletion of the PGKneo cassette from the Sox2^ENHneo allele was diagnosed by PCR (see above) and confirmed by Southern analysis with both a neo probe and probe (not shown).

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