Figure 5. Induction of germ line mutations with TALENs.

(A) TALEN RNA-injected G0 founders transmit targeted mutations to their offspring. G0 founders were mated with WT partners and the gDNAs of individual 1 dpf F1 progeny were examined by HRMA for the presence of mutations. The region flanking targeted sites was cloned and sequenced following PCR amplification from the gDNA of F1 offspring. Examples of germ line-transmitted mutations at each targeted locus are shown (purple and pink boxed sequences indicate Left and Right RVD binding sites of the TALENs, respectively; red indicates sequence alterations). (B, C) The germ lines of individual G0 founders are mosaic and can harbor several targeted mutations. Sibling F1 progeny of a single G0 founder targeted at gol (B) or a single G0 founder targeted at ryr3 (C) were examined by HRMA for inheritance of new mutations at gol or ryr3, respectively. Some F1 embryos (+/+) had no mutations, as their HRMA profiles (blue) resembled that of control WT embryos (grey). Other F1 embryos (m/+) displayed HRMA profiles indicating heterozygosity for a newly induced mutant allele (red). Several distinct HRMA profiles were identified among the heterozygous F1 progeny of a single G0 founder, indicating multiple mutations were transmitted by each G0 founder.