Fig. 2

Generation of mstn knockout yellow catfish using engineered zinc finger nucleases.

(A) 7 yellow catfish founders were identified to potentially carry mutated mstn in their germ cells by PCR. Agarose gel electrophoresis revealed that embryos from 7 yellow catfish founders gave out a predicted 186 bp product. The results suggested these fish carried a mutated mstn with the 4 bp insertion in their germ cells. “Wild type” and “Positive” denote the templates used for the 1^st round PCR were the genomic DNA isolated from a wild type yellow catfish (negative control) or the mutated mstn molecules with the 4 bp insertion (positive control), respectively. “July21-1, July21-6, July22-3, July22-5, July22-6, July29-7 and July29-8” denote the templates used for the 1^st PCR were the genomic DNA isolated from the different founders, respectively. (B) Sequencing chromatography shows the PCR products from two juvenile yellow catfish containing different mutated mstn alleles in their genomes. Arrow heads indicate that the mutated mstn alleles have different sequences from wild type allele stating from the base pointed. (C) Schematic diagram shows two mutated proteins would be produced from the two different strains of yellow catfish carrying different mutated mstn alleles. Mstn: Yellow catfish wild type Mstn comprises a signal sequence (N-terminal secretory signal), a propeptide domain (propeptide) and a bioactive domain (C-terminal domain). RX is a proteolytic site to remove the signal sequence and RXXR is a proteolytic processing site (RSSR) to produce bioactive form of Mstn. The number shows the position of amino acid residue [4]. Mstn^nju6: A truncated protein encoded by mstn^nju6 contains only the 42 amino acid residues of the N-terminal secretory signal of yellow catfish Mstn plus 5 new amino acid residues. Mstn^nju7: A mutated protein encoded by mstn^nju7 lacks two amino acid residues between aa37 and aa40 in the N-terminal secretory signal of yellow catfish Mstn.