Group II intron-stimulated homologous recombination in <i>X. laevis</i> oocyte nuclei with RNPs containing lariat or linear intron RNA and wild-type or RT-deficient intron-encoded protein.

Target plasmid pBRR3-ltrB (4.1 ng) and linear donor DNA (17 ng) were injected together into X. laevis oocyte nuclei in 16 nl of a solution containing 500 mM MgCl2, followed within 1 min by injection of Ll.LtrB RNPs (38 ng, 20 nl). RNPs were reconstituted with Ll.LtrB lariat or linear intron RNA without a T7 promoter in DIV and wild-type or RT-deficient mutant LtrA protein (RT−). After incubating for 2 h at 25°C, nucleic acids were extracted from 10 pooled oocytes for each condition and electroporated into E. coli HMS174(DE3). Targeting efficiencies were determined by the ratio of (TetR+AmpR)/AmpR colonies, as in intron-integration assays (Fig. 1 and Materials and Methods). The experiment was repeated three times with similar results.