Fig. 1.
Generation and genotype of β3-/- mutant zebrafish. (A) A flowchart depicting the generation of a zebrafish germline mutation. CRISPR guide RNA (gRNA) and Cas9 mRNA was injected into one-cell stage embryos. The injected embryos were raised and outcrossed with wild-type (WT) to generate heterozygous F1 fish. Mutant fish were identified by fluorescence polymerase chain reaction and Sanger sequencing. F1 siblings carrying the same mutation were then crossed to generate F2 progeny, and phenotype–genotype correlations were done using F2 embryos. (B) DNA sequencing at the target sequence revealed a 10-bp insertion that is predicted by in silico translation to cause a frameshift and a premature stop codon (underlined sequence) downstream of CRISPR guide RNA target site, truncating the protein within the extracellular domain (*). (C) Real-time quantitative polymerase chain reaction revealed that GABRB3 mRNA in homozygotes was significantly degraded. Bar graphs summarize normalized results (mean ± SD) of triplicate measures with 15 pooled larvae per group. (D) Examples of current traces recorded from voltage-clamped oocytes exposed to 100 µM etomidate (ETO; application indicated by bars over traces). Oocytes injected with WT zebrafish (z-fish) β3 mRNA produce large currents, while oocytes injected with mRNA derived from the mutated β3-/- gene produce no current (note tenfold amplified current scale). Traces from an oocyte injected with human β3 mRNA and from an uninjected oocyte are shown for comparison. (E) Columns represent the ratios of voltage-clamp currents elicited with 100 µM propofol (IPRO) and 100 µM etomidate (IETO) recorded in the same oocyte expressing either zebrafish or human β3 subunits (n = 5 each). The P value is based on unpaired two-tailed Student’s t test.