Zebrafish AhR2 Mutant Generation

Adult zebrafish were housed according to Institutional Animal Care and Use Committee protocols at Oregon State University on a recirculating system with water temperature of 28±1°C and a 14 h light/10 h dark schedule. Zebrafish embryos carrying a point mutation in ahr2 (ahr2^hu3335 strain) were requested and generously provided by the Hubrecht Institute. The ahr2^hu3335 line was identified from a library of N-ethyl-N-nitrosourea (ENU)-mutagenized zebrafish using the TILLING method as previously described [51] (link). Offspring of heterozygous ahr2^hu3335 carriers were raised to adulthood at the Sinnhuber Aquatic Research Laboratory, and genotyped for the ahr2^hu3335 point mutation with DNA isolated from fin clips [51] (link). PCR amplification was performed with genomic DNA and ahr2 gene-specific primers (Table 4), the product was purified using a QIAquick PCR purification kit (Qiagen) and sequenced with an ABI 3730 capillary sequencer at the Center for Genome Research and Biocomputing at Oregon State University. Homozygous carriers of the T to A point mutation in residue 534 (Figure 1) were identified to create an ahr2^hu3335 population. Because the TILLING method relies on random mutagenesis, mutant lines of interest carry other mutations throughout the genome. F1 fish are predicted to carry 3–6000 mutations and multiple outcrosses are necessary to reduce off-target mutations [52] (link). ahr2^hu3335 carriers were outcrossed to the wild type 5D (ahr2^+/+) line, and homozygous mutants were identified from an incross of their progeny. The ahr2^hu3335 mutant line has been maintained with subsequent outcrosses on the wild type 5D background, which was also used for all ahr2⁺ control experiments in our laboratory.
All developmental toxicity experiments were conducted with fertilized embryos obtained from group spawns of adult zebrafish as described previously [53] (link). Embryos used in experiments are defined as homozygous (ahr2^hu3335), heterozygous (ahr2^hu3335/+) or wild-type (ahr2⁺) for the point mutation in AHR2.