Zebrafish is an excellent animal model for target validation because of its genetic and physiological similarity to mammals. Its transparency enables thorough assessment of gene expression patterns and effects of loss or gain of gene function. Phylonix’s target validation service includes gene knockdown using chemically-modified antisense oligos, gene expression pattern analysis using whole mount in situ hybridization, and ectopic gene expression using mRNA injection. We also perform RT-PCR and 5’-RACE to clone zebrafish homologues of the genes of interest to design and generate antisense oligos, probes, and mRNA.

Gene knockdown: Antisense oligos which bind to RNA are injected into zebrafish at the one cell stage, resulting in uniform distribution of the oligos in the zebrafish. The effects of gene knockdown can be analyzed by a number of different assays, including visual assessment, dye labeling, antibody staining, and whole mount in situ hybridization.

Target validation using antisense RNA based-gene knockdown. (A) control. (B) zebrafish injected with fluorescein-tagged antisense oligos targeting the VEGF gene. The blood circulatory system was labeled in red by microangiography. The green fluorescence in (B) indicates uniform distribution of the oligos. (C) and (D) are high magnification pictures showing the blood vessels in the trunk region of a control zebrafish (C) and a VEGF antisense oligo injected zebrafish (D). Consistent with the results in mammals, knocking down the VEGF gene resulted in disruption of angiogenesis. The intersegmental vessels (ISVs), which are generated by angiogenesis, were severely disrupted in zebrafish injected with VEGF antisense oligo. Arrows in (A) and (C) point to normal ISVs in control zebrafish. Arrows in (B) and (D) point to disrupted or absent ISVs in VEGF antisense oligo injected zebrafish.

Ectopic expression of a mutated gene in zebrafish. Ectopic expression of a mutated gene can interfere with normal functions of an endogenous gene and can be used to assess target validation. In vitro synthesized RNAs or a DNA vector can be injected into zebrafish at different stages to inhibit gene function. For example, we injected the mutated gene responsible for ALS (Amyotrophic lateral sclerosis; Lou Gehring’s disease), G93A SOD (superoxide dismutase), into zebrafish and observed defective motorneurons in G93A injected zebrafish. (A) control zebrafish exhibit organized motor neurons; (B) G93-injected zebrafish exhibit disrupted motor neurons.

In situ hybridization: Zebrafish are labeled with sense or antisense probes using standard whole mount in situ hybridization procedure.

Whole mount in situ hybridization showing the expression pattern of the Desmin gene. Desmin, which encodes a muscle-specific class III intermediate filament, is expressed in the somites (arrow).