Figure 2

Workflow for establishing gene knockout Xenopus lines by TALENs. After selection of TALENs target site and assembly of TALEN plasmids, mRNAs encoding a pair of TALENs were microinjected into Xenopus embryos at one cell stage. The gene disruption efficiency was examined by PCR-based assay and afterwards sequencing. Some of the injected embryos were raised till sexual maturity. Individual G0 founder frog was mated with WT frog for germ line transmission to get F1 frogs. The F1 frogs were genotyped before metamorphosis by cutting tail and were raised to adulthood for intercross. Theoretically, ~25% offspring from F1 Xenopus will be homozygotes frogs carrying a specific gene mutation.