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Fig. 1
In vivo recombination analysis. (A) Overview of HR detection system by co-injection of TALEN RNAs and a GFP-inserted targeting construct in zebrafish embryos. (B) TALEN target in the sox2 locus and a sox2-targeting construct. Black, gray and blue blocks represent ORF, 5′ UTR and 3′ UTR, respectively (sox2 has no introns). G (0 position, marked in red) just before the stop codon is duplicated in the targeting construct locus and the 2a-sfGFP fragment is inserted in between the Gs. (C) sox2 TALEN RNAs were injected without any donor DNA and its mutagenic activity was measured by PCR and restriction enzyme analysis. Each lane represents an NdeI-digested amplicon encompassing sox2 TALEN target sequence from genomic DNA isolated from five embryos. The NdeI-digested wild-type PCR products are 78 and 75bp. (D-F) Lateral (D) and anterior-dorsal (E,F) views of 2dpf wild-type embryo. Box marks dorsal diencephalon region. (F) Detection of sox2 transcripts by whole-mount in situ hybridization. Scale bar: 200µm. (G,H) Confocal microscope images of the brain region that is similar to that shown in the boxed areas in D-F. (G) An embryo injected with a circular form of a sox2 reporter targeting construct. (H) An embryo co-injected with circular form of the construct and sox2 TALEN RNAs. Groups of cells expressing sfGFP in the diencephalon region are indicated by arrows. Scale bar: 20µm.