19Proc. Arthropod. Embryol. Soc. Jpn. 49, 19–20 (2016)Ⓒ 2016 Arthropodan Embryological Society of Japan
ISSN 1341–1527

Gene Knockout by Transcription Activator-like Effector Nucleases(TALENs) in the Sawfly, Athalia rosae ruficornis Jakovlev(Hymenoptera)*
Masatsugu HATAKEYAMA1) and Megumi SUMITANI2)
Division of Insect Sciences, National Institute of Agrobiological Sciences, Owashi Tsukuba, Ibaraki 305–8634, Japan
Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305–8634,
Japan
E-mail: sawfly@affrc.go.jp (MH)1)
2)

Recent progress in genomics and transcriptomics
together with the establishment of new molecular tools
enables questions in evolution, development and physiology
to be addressed at the molecular level. Experimental methods
inducing gene misexpression, such as transgenesis, geneknockdown and knockout, contribute to accelerate genefunctional analysis in insects (Daimon et al., 2014). In thesawfly, Athalia rosae ruficornis, transposon (piggyBac)mediated germline transformation and gene silencing(knockdown) by RNA interference have been established(Sumitani et al., 2003, 2005; Yoshiyama et al., 2013). Here, weshow that gene disruption (knockout) is feasible in thisspecies by using transcription activator-like effector
nucleases (TALENs), one of the genome editing tools used ina variety of organisms (Joung and Sander, 2013). A pair ofTALENs consist of a DNA-binding protein derived from plant
pathogenic bacteria fused to the DNA cleavage domain of arestriction endonuclease FokI, and FokI dimer createstargeted double-strand breaks, resulting in an insertion/deletion mutation upon repair of the break (Christian et al.,2010; Miller et al., 2011).
In the present study, TALENs were designed using ahigh efficient construct developed by Cermak et al. (2011) andTakasu et al. (2013), to bind specifically to the sequences
corresponding to the chromophore region of the enhanced
green fluorescent protein (EGFP) gene. These TALENcoding DNA fragments were transcribed to mRNAs in vitro
and injected into the anterior end of mature unfertilized eggs
taken from the transgenic strain carrying an EGFP transgene
driven by the 3xP3 eye-specific promoter (Hatakeyama et al.,2009). More than a half of the G0 males (26/41) showed achimeric phenotype in terms of eye EGFP expression,suggesting that cells bearing the disrupted EGFP gene
contributed to development. After progeny testing, four out of
26 chimeric G0 males produced EGFP-negative G1 females. As
these G1 females retained the EGFP transgene with deletionby some base pairs at the targeted region, we concluded thatthese females were originated from EGFP-disrupted sperm of
the chimeric G0 males and, hence, the EGFP-knockout
individuals.
By contrast, about 15% of the G0 males (8/53) showed achimeric phenotype when the TALEN-mRNAs were injected
at the posterior end of the eggs. Six out of eight chimeric G0
males produced presumable EGFP-knockout G1 females. Itremains to confirm insertion/deletion in the EGFP transgeneof these G1 females. We nevertheless, conclude that geneknockout by TALENs is feasible in A. rosae ruficornis.
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