TALENs were assembled using the Golden Gate method [50 (link)]. For targeting the mt2 locus, a 5′ RVD (NH–NG–NH–NH–NI–NG–NI–HD–NG–HD–NG–HD–NG–NH (DNA sequence GTGGATACTCTCTGG)) and a 3′ RVD (NI–HD–NG–HD–NG–NG–NH–NH–HD–NI–HD–NI–NG–NG (DNA sequence ACTCTTGGCACATTC)) were generated with a spacer of 16 bp (AAAAATGGACCCCTGC) to target exon 1. An AvaII (New England BioLabs) restriction site within the spacer region was used for genotyping of putative founders. mRNA was generated using the T3 mMessage mMachine Kit (Ambion) and injected using 100 pg of the TALEN mix.
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