Standard primer for SOMA-construction of a GFP reporter (A).

<p>Phosphorylated primer (red P) with diagnostic EcoRI site (red C; EcoRI*), 23 bp of GFP (green sequence) and morpholino target sequence in frame with GFP (NNN)_x including ATG (red sequence). Green fluorescent protein (GFP) gene (green); ampicillin resistance gene (Amp^R; yellow); <i>URA3</i> gene, yeast centromere (<i>CEN</i>), autonomous replication sequence (<i>ARS</i>) (red). Schematic outline of the SOMA method for production of GFP reporters (B). (1) denature pTP218; (2) 5′ phosphorylated single primer anneals; (3) primer is extended to the 5′ end; (4) nicks are ligated; (5) template strand is digested with DpnI; (6) mutated single stranded plasmid transformed into <i>E. coli</i> and positive clones identified by lack of EcoRI site. Test of TRIM2 GFP reporter construct <i>in vivo</i> (C). Synthetic reporter sense RNA injected into fertilized <i>Xenopus laevis</i> embryos at the one-cell stage; TRIM2- or Standard (STRD)-morpholino injected into one blastomere at the two-cell stage. Visualization after 1 day by brightfield or fluorescence microscopy (GFP). 99 of total 101 standard morpholino (STRD) injected embryos showed a GFP signal on both sides (98%); 249 of total 271 Trim2- morpholino injected embryos showed a GFP signal on one side (92%) (right).