Generating Zebrafish bag3 Mutants Using TALEN

TALEN techniques were employed to generate bag3 mutants, according to our previously published approaches (Shih et al., 2016 (link)). Briefly, TALEN primer pairs targeting the 2nd exon of the bag3 gene were designed using Zifit (http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx). The TALEN left-arm binding sequence 5′-TGTCATGAAAACCCTGAA-3′ and right-arm binding sequence 5′-ATCCTAGTTTCTCCTACA-3′ were used. Both TALENs were then assembled using a Golden Gate kit (Addgene) (Cermak et al., 2011 (link)). Capped mRNAs were synthesized using a mMESSAGE mMachine T3 kit (Ambion). Approximately 25 pg capped mRNA was injected into one-cell-stage embryos. Founder fish (F0) were raised to adulthood and outcrossed to generate F1 embryos. Individual F1 embryos were used for genotyping PCR to identify mutant alleles (forward primer: 5′-CGGCGTATAAAGAATTGCTGG-3′; reverse primer: 5′-GTGAAGTAGGTGAGCAAGAC-3′). The resulting PCR products were digested with the restriction enzyme PstI to identify the WT or mutant genotype. The uncut PCR products were Sanger-sequenced to determine the precise genomic lesions. Four different bag3 mutant alleles that presumably resulted in different shifts of the reading frame for each mutant locus were selected for continuous outcrosses up to the 5th generation and subsequent phenotypic analysis.