Direct gene disruption by TALENs in medaka embryos

• Procedures and key parameters of TALEN-mediated gene disruption were established.
• Gene dnd was efficiently disrupted by TALEN in medaka.
• Direct gene disruption by TALEN is germline transmittable and highly mosaic.
• Genomic dnd is dispensable during the post-meiotic haploid phase.

Targeted gene disruption (GD) is powerful for generating genetic alterations in animal genomes. Engineered endonucleases such as zinc finger nucleases and transcription activator-like effector nucleases (TALENs) allow for GD directly in animal embryos to achieve germline transmission. Here we report procedures and parameters of TALEN-mediated GD in the fish medaka by using a germ cell-specific gene dnd as a model. Embryos at the 1-cell stage were microinjected with synthetic TALEN mRNAs and examined for the survival rate and GD efficiency. Medaka embryos can tolerate a high dosage of TALEN-mRNA injection and exhibit a steadily increasing GD efficiency with increasing mRNA dosages before peaking at 100 ng/μl. This dosage produced ~ 24% efficiency for somatic GD. Some of the animals from manipulated embryos developed into fertile female and male. Most importantly, four fish (3 males and 1 female) examined by progeny-test were able to produce GD-bearing male and female gametes for germline transmission to F1 generation at ~ 10% efficiency. Therefore, TALEN is proficient for somatic and germline GD in medaka embryos, and disruption of one dnd copy does not compromise somatic development and gamete production.