Plasmid assays for site-specific group II intron integration in <i>D. melanogaster</i> and zebrafish embryos.

Site-specific integration of group II intron RNPs into a plasmid target site in D. melanogaster and zebrafish embryos was assayed as described in Figure 1 and Materials and Methods. In the D. melanogaster experiment, target plasmid (TP) with or without MgCl2 and Ll.LtrB lariat RNPs were injected separately into the posterior of 100 precellular blastoderm stage embryos, which were then incubated for 1 h at 30°C. In the zebrafish experiment, target plasmid with or without MgCl2 and Ll.LtrB lariat RNPs were injected separately into the cytoplasm of 10 one-cell embryos and incubated for 1 h at 30°C. After incubation, nucleic acids were isolated and electroporated into E. coli HMS174(DE3), and the integration efficiency was determined as the ratio of (TetR+AmpR)/AmpR colonies. Qualitatively similar results were obtained in two independent repeats of the Drosophila experiment with different batches of RNPs (integration efficiencies 1.2 and 1.8% with 0.25 M or 0.5 M MgCl2 and zero without MgCl2) and in three independent repeats of the zebrafish experiment (integration efficiencies 0.56, 0.63, and 0.82% with 0.125 M MgCl2 and 0 to 0.004% without MgCl2).