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Figure 1


Fig. 1. Targeted replacement of Sox10 by Sox8 in mice. (A) Schematic representation from top to bottom of the targeting construct, the Sox10 wild-type locus and the mutant locus before and after Cre recombination. The Sox10 exons (I-V) and the Sox8 open reading frame (ORF) are shown as boxes, 4.5 kb and 1.5 kb flanking regions as bars. Regions of homology between wild-type locus and targeting vector are depicted as black bars, introns 3 and 4 as open bars and surrounding genomic regions not contained in the targeting construct as stippled bars. Plasmid backbone sequences of the targeting construct are indicated by a thin line. Restriction sites for NcoI (N), BamHI (H) and ScaI (S) are shown, as well as the localization of 5' and 3' probes and the start codon of the Sox10 gene (ATG). The arrowheads indicate the localization of primers A-D used for quantitative RT-PCR. IRES-EGFP, EGFP open reading frame with preceding internal ribosomal entry site; neo, neomycin resistance cassette; loxP, recognition sites for Cre recombinase; Tk, Herpes simplex virus thymidine kinase gene cassette. (B) Southern blot analysis of DNA from wild-type (wt) and heterozygous (+/ki) ES cells 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 and 4.6 kb) and the targeted allele (6.0 and 5.4 kb) are indicated. (C) Southern blot analysis of DNA from wild-type (wt), heterozygous (+/ki) and homozygous (ki/ki) mice digested with NcoI for use of the 5' probe. (D) PCR analysis of Cre-mediated deletion of the neomycin resistance cassette in mice carrying a Sox10Sox8ki allele (+/ki and ki/ki). ES cells with a Sox10Sox8ki allele (+/ki) still contained the neomycin resistance cassette and served as control. M, size marker. (E) PCR genotyping of wild-type (wt), heterozygous (+/ki) and homozygous (ki/ki) mice. DNA fragments in the size marker (M) are 1.0 kb and 0.5 kb.





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