Figure 1

(A) Schematic of gnptab gene shows the locations of the left and right TALEN arms, the PCR primers used for genotyping (black arrowheads), and 3 isolated zebrafish lines carrying 2, 5, and 7 bp frame-shifting deletions. The predicted “translation” of these products is listed. (B) BslI digestion of genomic DNA identifies gnptab WT (+/+), heterozygous (+/–), and homozygous mutant (–/–, MLII) animals. (C) High-resolution melt analyses yields unique patterns that confirm the 3 expected genotypes. (D) Schematic illustrates live embryo dissections used in HRM analyses to assign the genotypes before experiments. Images of 3- and 5-dpf-old WT and MLII (gnptab^–/–) animals from lines carrying 5 and 7 bp deletions (gnptab^ga2.5 and gnptab^ga3.7) show progressive cardiac edema. Percent values equal the number of embryos exhibiting phenotypes similar to the picture. n = 50–60 embryos from 4–5 independent matings per line. Scale bar: 100 μm. Red arrowheads indicate edema; black arrowhead indicates pooled blood. (E) RT-PCR analyses of gnptab expression of embryos from 2 pools of WT and 3 pools of 5 bp–deleted and 7 bp–deleted embryos show reduced transcript abundance. Analyses of rpl4 transcripts provides an internal reference. Representative gel of 4 independent experiments. (F) Quantitation of transcript abundance show 60%–75% reduction in MLII lines from 3 to 5 dpf. Gel extraction and sequencing show 100% of residual transcripts in the mutant lines are mutant mRNAs. n = 100 embryos from 4 experiments, with 20 cloned transcripts sequenced per condition. ***P < 0.001, Dunnett’s test with correction.