Figure 3

Figure 3

Evaluation of conditional manipulation of paired positive and negative conditional Bi-FoRe KI alleles at the sox10 locus. (A) Schematic diagram of the positive conditional allele (Forward KI allele) sox10^{Bi−66-FoR-ReG−71} before and after Cre-mediated recombination. (B) Z-stack confocal images of F₂ embryos obtained from incross of F₁sox10^{+/Bi−66-FoR-ReG−71} heterozygotes. Upper panel: An embryo without Cre mRNA injection, showing tdTomato expression and normal pigmentation. Lower panel: An embryo after Cre mRNA injection, showing EGFP expression and defects in pigmentation, resembling sox10 mutant phenotype. Scale bar, 200 μm. (C) Junction PCR results of the F₂ embryos from incross of sox10^{+/Bi−66-FoR-ReG−71} after Cre mRNA injection. EGFP⁺: pooled genomic DNA template of the F₂ progeny showing EGFP expression after Cre mRNA injection. WT: pooled genomic DNA template of wild-type embryos. (D) RT-PCR results using the cDNA of the F₂ embryos from B with (Knockout) or without (Control) Cre mRNA injection. (E) Schematic diagram of the negative conditional alleles (Reverse KI allele) sox10^{Bi−71-ReG-FoR−66−1} and sox10^{Bi−71-ReG-FoR−2} before and after Cre-mediated recombination. (F) Z-stack confocal images of the embryos obtained from a cross of F₀ #1 bearing the sox10^{Bi−71-ReG-FoR−66−1} allele with #7 bearing the sox10^{Bi−71-ReG-FoR−66−2} allele. Upper panel: F₁ embryo without Cre mRNA injection, showing EGFP expression as well as defects in pigmentation, resembling sox10 mutant phenotype. Lower panel: F₁ embryo with Cre mRNA injection, showing tdTomato expression and recovery of pigmentation. Scale bar, 200 μm. (G) Junction PCR results of the F₁ embryos from the cross of F₀ #1 with #7 after Cre mRNA injection. tdTo⁺: pooled genomic DNA template from the F₁ progeny showing tdTomato expression after Cre mRNA injection. WT: pooled genomic DNA template of wild-type embryos. (H) RT-PCR results using the cDNA of the embryos from panel F with (Rescue) or without (Defective control) Cre mRNA injection. The location of RT-PCR primers in D and H is indicated in Fig. S3C. The expected size of the band is 467 bp