Fig. 4

Analysis of sox2-2a-sfGFP knock-in lines. (A) Schema of HR-mediated sfGFP insertion. Either NotI (or NaeI) or NcoI-digested targeting constructs (M1, M2, M3 and S1, see Fig. 2A) were co-injected with sox2 TALEN RNAs. Red bar indicates the probe sequences used for Southern blot. (B) Southern blot analysis. PstI/BamHI double-digested genomic DNA from individual adult fish was hybridized with the probe (red bars in A). Expected fragment size: wild type, 6.5kb; targeted, 7.3kb. Arrowheads indicate bands of unexpected size (lane 8, ~10kb; lane 9, ~14kb; lane 10, ~10kb). (C-G) PCR genotyping analysis. Primer pair locations are shown in A. WT, wild type; 1-12, sox2-2a-sfGFP F1 lines (see Table 1). (C) Detection of sfGFP (sF1+sR1 primers). (D) Amplicon extended from 5′ of the LA to within the sfGFP sequences (sF2+sR2). (E) Amplicon spanned the entire RA sequences (sF3+sR3). Arrowhead indicates a fragment of aberrant size (7.5kb). (F) Amplicon extended from 5′ of the LA to 3′ of the RA sequences (sF2+sR3). Expected amplicon size: wild type, 3.5kb; targeted, 4.3kb. (G) Amplicon for vector-specific sequences (F4+R4). (H) Quantitative PCR analysis of sfGFP relative copy numbers. Data are mean±s.e.m. (I,J) Lateral views of 2dpf embryos. (I) sox2 expression in wild type. (J) sfGFP expression in sox2-2a-sfGFP^stl84. Arrows indicate neuromasts. Scale bars: 200µm. (K-N) Transverse spinal cord section of 7 dpf sox2-2a-sfGFP^stl84 larva. (K) sfGFP expression. (L) Anti-HuC/D antibody staining. (M) Anti-Sox10 antibody staining. (N) Merged image of K-M. Brackets indicate sfGFP-positive cell clusters in posterior median sulcus (top) and posterior median septum (bottom). Arrows indicate sfGFP-positive and Sox10-positive OPCs. Arrowheads indicate sfGFP-positive and HuC/D-positive neurons. Scale bar: 20µm.