In vitro-transcribed RNA and Morpholino Microinjection in Zebrafish

In vitro-transcribed RNA of wild type Tnfa and DN Tnfr2 [15 (link)] was obtained following manufacturer’s instructions (mMESSAGE mMACHINE kit, Ambion). Morpholinos were diluted in DEPC-treated water at a concentration of 0.3 mM (Standard-mo, Gene Tools) 0.5 mM (Tnfa-MO, 5’-GCAGGATTTTCACCTTATGGAGCGT-3’ [42 (link)], 0.65 mM (Tnfr1-mo, 5’-ctgcattgtgacttacttatcgcac-3’ [15 (link)], 0.3 mM (Tnfr2-mo, 5’-ggaatctgtgaacacaaagggacaa-3’ [15 (link)]. Morpholinos and RNA were mixed in microinjection buffer and microinjected into the yolk sac of one-cell-stage embryos using a microinjector (Narishige) (0.5–1 nl per embryo). The same amount of MOs and/or RNA were used in all experimental groups. The efficiency of the MOs was checked by RT-PCR [15 (link),42 (link)].