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Knockout of a transgene by transcription activator‐like effector nucleases (TALENs) in the sawfly, A thalia rosae (Hymenoptera) and the ladybird beetle, H armonia axyridis (Coleoptera)
Author(s) -
Hatakeyama M.,
Yatomi J.,
Sumitani M.,
Takasu Y.,
Sekiné K.,
Niimi T.,
Sezutsu H.
Publication year - 2016
Publication title -
insect molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.955
H-Index - 93
eISSN - 1365-2583
pISSN - 0962-1075
DOI - 10.1111/imb.12195
Subject(s) - biology , transcription activator like effector nuclease , transgene , genetics , gene knockout , green fluorescent protein , gene , microbiology and biotechnology , genome , genome editing
Transcription activator‐like effector nucleases (TALENs) are efficient tools for targeted genome editing and have been utilized in a number of insects. Here, we demonstrate the gene disruption (knockout) caused by TALENs targeting a transgene, 3xP3 ‐driven enhanced green fluorescence protein ( EGFP ), that is integrated in the genome of two species, the sawfly Athalia rosae (Hymenoptera) and the ladybird beetle Harmonia axyridis (Coleoptera). Messenger RNAs of TALENs targeting the sequences adjacent to the chromophore region were microinjected into the eggs/embryos of each species. In At. rosae , when microinjection was performed at the posterior end of eggs, 15% of G 0 individuals showed a somatic mosaic phenotype for eye EGFP fluorescence. Three‐quarters of the somatic mosaics produced EGFP‐negative G 1 progeny. When eggs were injected at the anterior end, 63% of the G 0 individuals showed somatic mosaicism, and 17% of them produced EGFP‐negative G 1 progeny. In H. axyridis , 25% of posterior‐injected and 8% of anterior‐injected G 0 individuals produced EGFP‐negative G 1 progeny. In both species, the EGFP‐negative progeny retained the EGFP gene, and various deletions were detected in the target sequences, indicating that gene disruption was successfully induced. Finally, for both species, 18–21% of G 0 founders produced gene knockout progeny sufficient for establishing knockout strains.