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Targeted indel mutation induced by engineered Cas9/gRNA at the cftr gene in zebrafish. (A) The target sites highlighted by yellow and the PAM sequence marked by red underlined text. Deletions of cftr mutant are shown as dashes. Boxes show the start codon of WT and destroyed start codon of cftr mutant. (B) Gel shows T7E1 digestion of PCR products amplified from adult tail genomic DNA of F1 heterozygous generation. The uncleaved and cleaved PCR products are indicated. After digestion with T7E1, the cleaved PCR product of the adult tail represents the fragments containing the mutation. (C) Sequencing results show that F1 heterozygous generation fish carrying cftr mutant produces overlapping peaks marked by dashed box. (D) Western blot assay indicates the significant reduced Cftr protein level in offspring embryos from mutant line. (E) The got genotyped homozygous cftr mutant embryo also demonstrated the absent of Kupffer’s vesicle (KV) lumen (pointed by arrow) at 8-somite stage.
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