Figure 3. Knockout of gas7 promotes NB metastasis in transgenic fish overexpressing MYCN.

(A) Schematic diagram of the targeted site for TALEN in exon 2 of gas7. TALEN binding sites are indicated by arrows in the nucleotide sequences alignment of wild-type (WT) and mutant gas7 (gas7mut) alleles. Red box marks the 7-base pair (bp) deletion (del) in the gas7 mutant.

(B) Alignment of amino acid sequence of WT and Gas7 mutant protein. The 7-bp deletion leads to a truncated protein product with short regions of altered translation (indicated in red) and pre-mature stop codon (*).

(C) Diagram of the predicted Gas7 mutant protein with loss of most of the F-BAR domain.

(D) Gel electrophoresis of PCR products from WT, gas7mut heterozygous (gas7mut het) or gas7mut homozygous (gas7mut homo) larvae with or without Fok I digestion. Intact bands at 546 bp were detected in gas7mut het or homo larvae after Fok I digestion, suggesting loss of Fok I cut sites in these mutants.

(E) Immunoblot analysis of protein lysates from 5-day old gas7mut het, gas7mut homo or WT larvae zebrafish, as well as in vitro synthesized Gas7 protein (Control). The expression levels of Gas7 are either decreased in the gas7mut het larvae or undetectable in the gas7mut homo larvae. Equal loading was confirmed by stripping the membrane and reprobing with antibody against Gapdh.

(F) Fraction of tumor-bearing fish that developed metastases at 18 weeks of age. Differences in the percentage of MYCN-only versus MYCN;gas7mut het or MYCN;gas7mut homo are significant by Fisher’s exact test at p=0.002 or p=0.0004, respectively.

(G-I) Fluorescence images of MYCN;gas7mut het (G) or MYCN;gas7mut homo compound fish (H), or MYCN-only transgenic fish (MYCN, I) with EGFP-expressing primary tumors in the interrenal gland regions or metastatic tumors (white arrows, G and H) at 18 weeks postfertilization (wpf). Scale bar, 1 mm.