Figure 1.
Group II intron-integration assay in X. laevis oocyte nuclei.
(A) Plasmid assay. Microinjected group II intron RNPs containing a 0.9-kb Ll.LtrB-ΔORF intron RNA with a T7 promoter in DIV and the group II intron RT integrate into a target site (ligated ltrB exon 1 and 2 sequences; E1 and E2) cloned upstream of a promoterless tetR gene in an AmpR target plasmid (pBRR3-ltrB), thereby activating the tetR gene. T1 and T2 are E. coli rrnB transcription terminators, and Tφ is a phage T7 transcription terminator. (B) Protocol. Target plasmids and RNPs were injected into oocyte nuclei using different needles. After incubating the oocytes for different times, nucleic acids were isolated and electroporated into E. coli HMS174(DE3). The integration efficiency was calculated as the ratio of (TetR+AmpR)/AmpR colonies.
Table 1.
Site-specific integration of a group II intron into a plasmid target site in X. laevis oocyte nuclei.
Figure 2.
Determination of optimal conditions for site-specific integration of group II intron RNPs into a plasmid target site in X. laevis oocyte nuclei.
(A) Mg2+-concentration dependence. Target plasmid DNA (10 ng) with 0 to 2,000 mM MgCl2 and 17 mM dNTPs was injected into X. laevis oocyte nuclei followed by Ll.LtrB lariat RNPs (50 ng). The oocytes were incubated for 2 h at 25°C. (B) Temperature dependence. Target plasmid DNA (9 ng) with 500 mM MgCl2 and 17 mM dNTPs was injected into X. laevis oocyte nuclei followed by Ll.LtrB lariat RNPs (50 ng). The oocytes were incubated for 2 h at different temperatures. (C and D) Time courses at 25°C or 30°C, respectively. Target plasmid DNA (4.5 ng) with 500 mM MgCl2 and 17 mM dNTPs was injected into X. laevis oocyte nuclei, followed by Ll.LtrB lariat RNPs (36 ng). The oocytes were incubated for the indicated times and then quick frozen on dry ice. Nucleic acids isolated from the 25°C-incubated oocytes were untreated or digested with KpnI or MluI (10 units; New England Biolabs) for 1 h at 37°C and then extracted twice with phenol-CIA and ethanol precipitated prior to electroporation into E. coli. In (A)–(D), injection volumes were 18 nl. Integration efficiencies (%) were determined by electroporating nucleic acids extracted from oocytes into E. coli HMS174(DE3), followed by plating to determine ratios of (TetR+AmpR)/AmpR colonies, as described in Figure 1 and Materials and Methods. Each experiment was repeated at least once with essentially the same results.
Figure 3.
Assay of group II intron-RNP stimulated homologous recombination in X. laevis oocyte nuclei.
A target plasmid (pBRR3-ltrB) containing the Ll.LtrB intron-insertion site (IS; ligated ltrB exon 1 and 2 sequences; E1 and E2) was co-injected into X. laevis oocyte nuclei with a 5.4-kb linear donor DNA, consisting of a 4-kb phage λ sequence with an inserted T7 promoter, flanked by 705- and 718-bp sequences homologous to those flanking the Ll.LtrB-insertion site in the target plasmid. Ll.LtrB RNPs containing the 0.9-kb Ll.LtrB-ΔORF intron and the group II intron RT were then injected into the same oocyte nuclei. A double-strand break resulting from intron RNA reverse splicing and second-strand cleavage at the Ll.LtrB target site stimulates homologous recombination, resulting in the insertion of the donor DNA containing the T7 promoter, thereby activating the tetR gene. Nucleic acids were isolated and electroporated into E. coli HMS174(DE3), and the targeting efficiency was calculated as the ratio of (TetR+AmpR)/AmpR colonies.
Table 2.
Group II intron-stimulated homologous recombination in X. laevis oocyte nuclei.
Figure 4.
Target DNA cleavage and target DNA-primed reverse transcription reactions of RNPs reconstituted with lariat or linear Ll.LtrB intron RNA.
Ll.LtrB RNPs containing lariat or linear intron RNA were incubated with 32P-labeled DNA oligonucleotide substrates containing the Ll.LtrB target site (positions −56 to +73 from the Ll.LtrB intron-insertion site), and the products were analyzed in a denaturing polyacrylamide gel. (A) Lariat and linear RNPs were incubated with internally labeled DNA substrate for 30 min at 37°C, as described in Materials and Methods. (B) and (C) Lariat and linear RNPs incubated with DNA substrates labeled (asterisk) at the 5′-end of the top and bottom strand, respectively in the presence of 0.2 mM dATP, dCTP, dGTP, and dTTP for 30 min at 37°C. In (A)–(C), products are indicated to the right of the gel. The schematic at the bottom diagrams the products expected for each reaction. All lanes are from the same gel, but some lanes in (A) were rearranged to appear adjacent.
Table 3.
Group II intron-stimulated homologous recombination in X. laevis oocyte nuclei with RNPs containing lariat or linear intron RNA and wild-type or RT-deficient intron-encoded protein.
Table 4.
Effect of chromatinization of the target plasmid on group II intron site-specific DNA integration and group II intron-stimulated homologous recombination in X. laevis oocyte nuclei.
Table 5.
Plasmid assays for site-specific group II intron integration in D. melanogaster and zebrafish embryos.
Figure 5.
Site-specific integration of retargeted Ll.LtrB introns into chromosomal target sites in the Drosophila melanogaster yellow (y) gene.
(A) DNA target site sequences and Ll.LtrB intron RNA base-pairing interactions for targetrons Y18a and Y3776s. Retargeted Ll.LtrB-ΔORF introns (targetrons) are denoted by a number that corresponds to the nucleotide position 5′ to the Ll.LtrB intron-insertion site numbered from the A of the ATG initiation codon, followed by “a” or “s”, indicating sense and antisense strand respectively. The DNA target sequences are shown from positions −30 to +15 from the intron-insertion site, with nucleotide residues that match those in the wild-type Ll.ltrB intron target sequence [7] highlighted in gray in the top strand. The intron-insertion site (IS) in the top strand and the IEP-cleavage site (CS) in the bottom strand are indicated by arrowheads. Below is shown a schematic of the Drosophila yellow (y) gene (NCBI accession number P09957), with the targetron-insertion sites indicated, and a diagram of the PCRs used to detect site-specific targetron insertion. y gene exons are gray rectangles, and introns and flanking sequences are lines. PCR primers used to detect and sequence the targetron integrations are indicated by numbered arrows. (B) PCR analysis of targetron Y18a integration using a double-injection protocol. Fifty embryos were injected with a solution containing 100 mM MgCl2+17 mM dNTPs followed by Y18a lariat RNPs (0.9 mg/ml). The embryos were incubated for 1 h at 30°C followed by 48 h at 18°C. Nucleic acids were isolated, and PCR products corresponding to the 5′- and 3′-junctions of Y18a integrated at its chromosomal target site (1,156-bp and 238-bp, respectively) were detected by nested PCR using the following primer pairs: 5′-junction, primers 1 (LtrB+940a) and 2 (yellow+277a), followed by primers 3 (LtrB+933a) and 4 (yellow+241a); 3′-junction, primers 5 (LtrB+788s) and 6 (yellow-350a), followed by primers 7 (LtrB+880s) and 8 (yellow-160s) (see Materials and Methods). (C) PCR analysis of targetron Y3776s integration using a double-injection protocol. Forty embryos were injected with 100 mM MgCl2 followed by Y3776s lariat RNPs (1.3 mg/ml), and the embryos were then incubated for 30 min at 37°C. Nucleic acids were isolated, and a 702-bp PCR product corresponding to the 3′-junction of Y3776s integrated at its target site was detected by PCR using primers 5 (LtrB+788s) and 9 (yellow+4325a) (see Materials and Methods). The dark band at the bottom of the gel is RNA. (D) PCR analysis of Y3776s integration using a single-injection protocol. Thirty embryos were injected with a solution containing100 mM KCl, 5 mM putrescine dihydrochloride, 3 mM spermidine trihydrochloride, 1 mM spermine tetrahydrochoride, 5 mM MgCl2, and Y3776s linear RNPs (0.5 mg/ml). After incubating the embryos for 30 min at 37°C, nucleic acids were isolated as described above, and a 348-bp PCR product (arrow) corresponding to the 3′-junction for Y3776s integrated at its target site was detected by nested PCR using primers 5 (LtrB+788s) and 9 (yellow+4325a), followed by primers 10 (LtrB+870s) and 11 (yellow+4054a) (see Materials and Methods). Other bands in the gel are likely due to non-specific annealing of the primers. In (B–D), the PCR products were analyzed by electrophoresis in a 1% agarose gel, which was stained with ethidium bromide, and the identities of the PCR products were confirmed by sequencing across the integration junctions (not shown).