Group II intron-stimulated homologous recombination in <i>X. laevis</i> oocyte nuclei.

Target plasmid pBRR3-ltrB (TP; 4.1 ng) and linear donor DNA (17 ng) were injected together into X. laevis oocyte nuclei in 17 nl of a solution containing 500 mM MgCl2, followed within 1 min by injection of Ll.LtrB RNPs (34 ng, 17 nl) reconstituted with Ll.LtrB lariat RNA without a T7 promoter in DIV. After a 2-h incubation at 25°C, nucleic acids were extracted from 10 pooled oocytes for each condition and electroporated into E. coli HMS174(DE3). In one sample, the target plasmid was precut with BtrI 27-bp upstream of the intron target site and injected without group II intron RNPs. Targeting frequencies were determined by the ratio of (TetR+AmpR)/AmpR colonies, as in intron-integration assays (Fig. 1 and Materials and Methods). Experiments for each condition except precut target plasmid DNA were repeated at least once with similar results.