Fig. S8

Injection of RNA encoding TALENs that target hspb7 generate cardiac defects that phenocopy the hspb7 morphants. A) Representative phenotypes of injected embryos at 3 dpf demonstrating cardiac morphogenetic defects including right-sided (left) or non-looping (right) abnormalities. B) Quantification of heart tube phenotypes at 30 hpf in wildtype (n = 29), or embryos injected with RNA encoding TAL1 alone (n = 58), TAL2 alone (n = 53), or TAL1+TAL2 (n = 57). C) Injection of TALEN RNA results in mutations at the hspb7 target site in embryos displaying cardiac morphogenetic defects when compared to uninjected controls. Lane 1) Amplicon from 5011/12 primer set using genomic DNA from uninjected embryos. Lane 2) Amplicon from lane 1 digested with HpyCH4V restriction enzyme. Lane 3) Amplicon from 5011/12 primer set using genomic DNA from Hspb7 TAL1+TAL2 injected embryos displaying cardiac morphogenetic defects. Lane 4) Amplicon from lane 3 digested with HpyCH4V restriction enzyme. The schematic above illustrates the PCR product that contains 3 potential HpyCH4V restriction sites (orange lines) one of which is in the TALEN-targeted sequence (red X). Unmodified DNA is digested to a 106/109 bp doublet, and a smaller 77 bp fragment (and a small 10 bp fragment that is not seen). Arrow indicates HpyCH4V resistant 183 bp fragment due to mutation of the HpyCH4V restriction site in the TALEN target region. Molecular size markers are in the leftmost lane.