Knockout of a transgene by transcription activator‐like effector nucleases (TALENs) in the sawfly, Athalia rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis (Coleoptera). Issue 1 (26th October 2015)

• Record Type: Journal Article
• Authors: Hatakeyama, M.; Yatomi, J.; Sumitani, M.; Takasu, Y.; Sekiné, K.; Niimi, T.; Sezutsu, H.
• Is Part Of: Insect molecular biology. Volume 25:Issue 1(2016:Feb.)
• Page Start: 24
• Page End: 31
• Publication Date: 2015-10-26
• Subjects: transcription activator‐like effector nucleases (TALENs) -- gene knockout -- Athalia rosae -- Harmonia axyridis -- EGFP transgene; Insects -- Molecular aspects -- Periodicals; 595.7
• DOI: 10.1111/imb.12195
• Languages: English
• ISSNs: 0962-1075

Abstract

Abstract: 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 G0 individuals showed a somatic mosaic phenotype for eye EGFP fluorescence. Three‐quarters of the somatic mosaics produced EGFP‐negative G1 progeny. When eggs were injected at the anterior end, 63% of the G0 individuals showed somatic mosaicism, and 17% of them produced EGFP‐negative G1 progeny. In H. axyridis, 25% of posterior‐injected and 8% of anterior‐injected G0 individuals produced EGFP‐negative G1 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 G0 founders produced gene knockout progeny sufficient for establishing knockout strains.