Transgenic Zebrafish with Z-AR Expression

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’.