Knockout of a transgene by transcription activator‐like effector nucleases (TALENs) in the sawfly, Athalia rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis (Coleoptera)

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),...

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Bibliographic Details
Published in:Insect molecular biology 2016-02, Vol.25 (1), p.24-31
Main Authors: Hatakeyama, M., Yatomi, J., Sumitani, M., Takasu, Y., Sekiné, K., Niimi, T., Sezutsu, H.
Format: Article
Language:English
Subjects:
Animals
Athalia rosae
Coleoptera
Coleoptera - genetics
EGFP transgene
Female
gene knockout
Gene Knockout Techniques
Green Fluorescent Proteins
Harmonia axyridis
Hymenoptera
Hymenoptera - genetics
Male
transcription activator-like effector nucleases (TALENs)
Transgenes
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Summary: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.
ISSN:0962-1075
1365-2583
DOI:10.1111/imb.12195