TALEN design and results of indel mutations in zebrafish <i>pak4</i>.

<p>(A) Alignment of wild type <i>pak4</i> sequence with mutated PCR amplicons. A pair of TALEN was designed overlapping (left arm) and downstream (right arm) the start ATG codon. The target sequences of the TAL Effector DNA-binding domains are highlighted in red. The start codon and two polymorphic nucleotides found within the right arm target are underlined. wt, wild type EK. Δ2, Δ4 and Δ5, alleles with 2-, 4- and 5-nucleotide deletion, respectively. Ins6, allele with 6-nucleotide insertion. (B) Results of immunoblotting for <i>pak4</i> protein. Total protein from 100 embryos at shield stage was collected, immunoprecipitated and analyzed using a custom-made zebrafish <i>pak4</i> anti-peptide antibody. wt, wild type EK. Δ2/Δ4, F₃ mutants of mixed genotypes <i>pak4</i>Δ2/Δ2, <i>pak4</i>Δ4/Δ4 and <i>pak4</i>Δ2/Δ4. <i>pak4</i> protein with a predicted molecular weight of 72 kDa was detected in the wild type embryos but not in the mutants. Duplicate membrane probed with antibody to α-tubulin was used as loading control.