Sp6 message rna polymerase kit

The avp morpholino oligonucleotide (MO) and mismatched-Avp MO were obtained from Gene Tools (Philomath, OR, USA). The sequences of MO were as follows: Avp MO, 5′-AGACAGCAGAGAGTCTGACATCTCG-3′; Avp-mismatched MO, 5′-AGACACCAGACAGTGTCACTTCTCG-3′. The MOs were prepared with 1× Danieau solution [58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO₄, 0.6 mM Ca(NO₃)₂, and 5.0 mM HEPES; pH 7.6]. MO solution containing 0.1% phenol red (for visualization) was injected into zebrafish embryos at the 1–2-cell stage using an IM-300 microinjector system (Narishigi Scientific Instrument Laboratory, Tokyo, Japan). For capped-mRNA (cRNA) injection, the corresponding Avp coding region was amplified by PRC and inserted into the pCS2⁺ vector. The construct was linearized with NotI, and cRNA was transcribed using an SP6 message RNA polymerase kit (Ambion, Huntington, UK). cRNA was injected into embryos at the 1- to 2-cell stage at 400 pg/embryo.

The zebrafish MR MO (5′-AAC TTTGGTATCTTTTAGTCTCCAT-3′) and GR MO, was designed against the gr splicing variant, (5′-CTGCTTCATGTATT TTAGGGTTCCG-3′) were designed as previously described (Lin et al., 2011 (link)). A standard control MO (5′-CCTCTTACCTC AGTTACAATTTATA-3′) was used as the control. The GR MO (3.5 ng/embryo) and MR MO (3.5 ng/embryo) were injected into embryos at the 1–2 cell stage using an IM-300 microinjector system (Narishige Scientific Instrument Laboratory, Tokyo, Japan). MO-injected embryos were sampled at 3 dpf for subsequent analyses. Rescue experiments for the defects caused by the GR MO were performed by synthesizing cRNA from a pCS2+ plasmid containing full-length GR. The expression plasmid was linearized and used as template for cRNA synthesis using an SP6 message RNA polymerase kit (Ambion, Austin, TX, USA). Finally, the full-length GR cRNA (300 pg/embryo) and GR were co-injected into embryos at the 1–2 cell stage, and embryos were sampled at 3 dpf.