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), 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.
Division of Insect Sciences, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Japan.
M
M
Hatakeyama
Laboratory of Sericulture & Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.
J
J
Yatomi
Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Japan.
M
M
Sumitani
Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Japan.
Y
Y
Takasu
Division of Insect Sciences, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Japan.
K
K
Sekiné
Laboratory of Sericulture & Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.
T
T
Niimi
Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Japan.
H
H
Sezutsu
D000818:Animals; D001517:Coleoptera / Q000235:genetics*; D005260:Female; D055786:Gene Knockout Techniques; D049452:Green Fluorescent Proteins; D006927:Hymenoptera / Q000235:genetics*; D008297:Male; D019076:Transgenes
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
Athalia rosae; EGFP transgene; Harmonia axyridis; gene knockout; transcription activator-like effector nucleases (TALENs)