The pair of TALENs targeting exon 2 of the cyp21a2 gene was generated with the Golden Gate TALEN Kit (Addgene, Cambridge, MA). TALEN target sites were determined by TAL Effector Nucleotide Targeter Version 2.0 (21 (link)). The sequences of the repeat-variable di-residues (RVDs) in the TAL Effector DNA-binding domains were RVD TALE1 (left): NG-HD-NG-NH-NH-NG-HD-HD-NG-HD-NH-HD-NG-HD-NG-HD and RVD TALE2 (right): NG-NH-NH-NH-HD-NH-NI-NH-NI-NG-HD-HD-NI-NH-HD-NI-NG-NH-NG. TALEN mRNA was synthesized with an SP6 polymerase mMessage mMachine Kit (Life Technologies, Waltham, MA) after 1 μg of plasmid DNA was digested with NotI.
TALENs were injected into one-cell-stage embryos, with 1 nL of injection solution containing 50 or 150 ng/μL of each TALEN diluted in nuclease-free water (Promega) plus 0.1% phenol red. F0 generations were grown to adulthood from the injected embryos and screened for transmission of cyp21a2 mutations. Identified founders were outcrossed to fish to generate F1 generations. The F1 generation fish were screened for heterozygous cyp21a2 mutations. F1 fish with defined cyp21a2 mutant alleles were outcrossed to their respective genetic backgrounds to generate the F2 generation. The heterozygous mutant fish of the F2 generation were incrossed to study cyp21a2 mutant phenotypes in embryos and larvae.
TALENs were injected into one-cell-stage embryos, with 1 nL of injection solution containing 50 or 150 ng/μL of each TALEN diluted in nuclease-free water (Promega) plus 0.1% phenol red. F0 generations were grown to adulthood from the injected embryos and screened for transmission of cyp21a2 mutations. Identified founders were outcrossed to fish to generate F1 generations. The F1 generation fish were screened for heterozygous cyp21a2 mutations. F1 fish with defined cyp21a2 mutant alleles were outcrossed to their respective genetic backgrounds to generate the F2 generation. The heterozygous mutant fish of the F2 generation were incrossed to study cyp21a2 mutant phenotypes in embryos and larvae.