Nanoject 2 injection apparatus

To produce Tg frogs, we used the I-SceI meganuclease- [18] (link) and transposon-mediated gene trap [19] (link). Fertilized eggs were injected with I-SceI meganuclease (NEB) and the I-SceI-cleaved plasmid encoding Z-AR and V5, and the Tol II mRNA [19] (link) and the prrT2ARG plasmid encoding Z-AR and GFP, respectively, using a NANOJECT II injection apparatus (Drummond). Tg embryos were cultured in 0.1×MMR with 6% Ficoll PM400 (GE Healthcare) and 50 µg/ml gentamicin (Wako), developed to St. 20 at 18°C, and then transferred to water at room temperature. The tadpoles were continuously reared in water with or without T (50 ng/ml; 150 nM). To confirm the exogenous Z-AR integration into genomic DNA, we extracted DNA from the tail tip of all Tg frogs just after metamorphosis, using the AllPrep DNA/RNA Micro Kit (QIAGEN). The PCR primers used were: forward, 5′-GCGGTTTTTCCAACTTACCA-3′ and reverse, 5′-CGAGACCGAGGAGAGGGTTA-3′).

We produced Tg ZW frogs, using the I-SceI meganuclease-mediated gene trap [3 (link)]. Fertilized eggs were injected with I-SceI meganuclease (NEB, Tokyo, Japan) and the I-SceI-cleaved plasmid encoding Z-AR and V5 (GKPIPNPLLGLDST), using a NANOJECT II injection apparatus (Drummond, Broomall, PA, USA). We also produced the placebo-Tg (pTg) ZW frogs as control by injecting I-SceI-cleaved pDPCG vectors and the meganuclease into ZW embryos. Tg and pTg embryos and tadpoles were reared as previously described [3 (link)]. To confirm Z-AR integration into genomic DNA, we extracted DNA from the tail tip of all Tg tadpoles at St. V, using the AllPrep DNA/RNA Micro Kit (QIAGEN, Tokyo, Japan) [10 ]. The PCR primers used were: forward, 5’-GCGGTTTTTCCAACTTACCA-3’ and reverse, 5’-CGAGACCGAGGAGAGGGTTA-3’.