Efficient knockout of transplanted green fluorescent protein gene in medaka using TALENs.

Abstract

Transcription activator-like effector nucleases (TALENs) are used for gene knockout and genome-editing studies in zebrafish, and these techniques have the potential to be applied to other fish species. Here, we show that TALENs can directly knock out a green fluorescent protein (GFP) transgene in medaka by affecting translation and synthesis of the GFP. We constructed a transgenic plasmid (pGFP-RFP) carrying the GFP and red fluorescent protein (RFP) genes, and used a modified TALEN method to assemble a pair of TALENs for the core chromophore Y66 region of GFP. Embryo toxicity of TALEN messenger RNA (mRNA) was far lower than the linearized plasmid; meanwhile, 76.3% embryos, green fluorescence of embryos decreased significantly after co-injection of TALEN mRNA and the linearized plasmid, but red fluorescence showed no significant change. Real-time quantitative polymerase chain reaction and sequencing results showed that nearly 100% mutated GFP position was disrupted at the Y66 region of GFP in the co-injected medaka embryos, caused by TALENs. This led to random insertion-deletion of nucleotides, which affected the translation of GFP and disrupted GFP synthesis. This provides new experimental evidence for designing TALEN sites in genes for which only key functional domains are known. Our results show that a modified TALEN method can efficiently and specifically mediate a transgene knockout in medaka. This report may promote the application of TALENs in gene-editing studies of fish species other than zebrafish.