Generation of a conditional Sox10 allele in mice. (A) Schematic representation, from top to bottom, of the targeting construct, the wild-type Sox10 allele (Sox10⁺), the conditional Sox10 allele before (Sox10^flneo) and after (Sox10^fl) removal of the neo selection cassette by Flp recombinase, and the deleted allele (Sox10^Δ) after Cre recombination. Sox10 exons (I–V) and the continuous Sox10 ORF used in the conditional allele are shown as boxes, and 4.5- and 1.5-kb-long flanking regions are shown as bars. Regions of homology between wild-type locus and targeting vector are depicted as black bars, introns 3 and 4 are depicted as open bars, and surrounding genomic regions not contained in the targeting construct are depicted as dashed bars. Plasmid backbone sequences of the targeting construct are indicated by a thin line. Restriction sites for NcoI (N), BamHI (B), and ScaI (S) are shown as well as the localization of 5′ and 3′ probes and the start codon of the Sox10 gene (ATG). neo, neomycin resistance cassette; FRT, recognition sites for Flp recombinase (depicted as ellipses); loxP, recognition sites for Cre recombinase (depicted as triangles); Tk, Herpes simplex virus thymidine kinase gene cassette. (B) Southern blot analysis of genomic DNA from Sox10^+/+ (wt) and Sox10^fl/fl (fl/fl) mice digested with NcoI for use of the 5′ probe and BamHI–ScaI for the 3′ probe. The size of bands corresponding to the wild-type (6.6 kb for the 5′ probe and 4.6 kb for the 3′ probe) and the targeted alleles (4.8 kb for the 5′ probe and 3.7 kb for the 3′ probe) are indicated. (C) PCR genotyping of Sox10^+/+ (wt), Sox10^+/fl (+/fl), and Sox10^fl/fl (fl/fl) mice. Size of DNA fragments in the marker (M) is indicated in kilobases on the left. (D) Whole mount GFP autofluorescence of Sox10^fl/fl embryos at 10.5 dpc. (E) Immunohistochemistry with antibodies directed against Sox10 on transverse sections from the thoracic region of Sox10^+/+ (wt) and Sox10^fl/fl (fl/fl) embryos at 12.5 dpc. (F) Comparison of expression levels for Sox10 in Sox10^+/+ (wt) and Sox10^fl/fl (fl/fl) embryos at 12.5 dpc with primers recognizing a common sequence in both transcripts using quantitative LightCycler RT-PCR. Transcript levels in each sample were normalized to Rpl8. After normalization, transcript levels in the wild type were arbitrarily set to 1, and transcript levels in Sox10^fl/fl are expressed relative to wild-type levels ± SEM. Experiments were repeated twice with material from two independently obtained embryos for each genotype and age. Bars: (D) 1 mm; (E) 100 µm.