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Bacteriophage phiC31 inserts its genome into that of its host bacterium via the integrase enzyme which catalyzes recombination between a phage attachment site (attP) and a bacterial attachment site (attB). Integrase requires no accessory factors, has a high efficiency of recombination, and does not need perfect sequence fidelity for recognition and recombination between these attachment sites. These imperfect attachment sites, or pseudo-attachment sites, are present in many organisms and have been used to insert transgenes in a variety of species. Here we describe the phiC31 integrase approach to make transgenic Xenopus laevis embryos.
Fig. 9.1.
Representation of φC31 integrase-mediated transgenesis in Xenopus laevis. The reporter plasmid containing an attB site and an insulated reporter gene is injected along with integrase mRNA into single-cell embryos. The chromosomes (thick black lines) contain numerous pseudo-attP sites. Inside the single-cell embryo, the integrase protein catalyzes recombination between the attB site in the reporter plasmid (thin black line) and a pseudo-attP site in the embryo’s genome (thick black line). Recombination results in the formation of two new attachment sites, attR and attL flanking the integrated reporter plasmid.
Fig. 9.2.
φC31 integrase-mediated transgenesis of insulated reporter plasmids generate Xenopus embryos with tissue-appropriate expression. In every case, the insert shows a brightfield image of the embryo. (A) Non-injected stage 42 embryo. (B) Stage 42 embryo injected with 5 pg of CMV-EGFP-DI-attB reporter plasmid. (C) Stage 42 embryo injected with 5 pg of CMV-EGFP-DI-attB reporter plasmid and 1 ng of integrase mRNA. (D) Stage 44 embryo injected with 5 pg of CL-EGFP-DI-attB plasmid alone. (E) Stage 44 embryo injected with 5 pg of CL-EGFP-DI-attB plasmid and 1 ng of integrase mRNA. GFP expression is indicated with the white arrow. (F) Southern blot demonstrating single integration events. Lanes 1 and 2 contains DNA harvested from single stage 46 CMV-EGFP-DI-attB transgenic embryos that expressed GFP uniformly. Lanes 3–4 contain DNA harvested from single stage 46 CL-EGFP-DI-attB transgenic embryos that expressed GFP in the lens of the eye. Lane 5 contains stage 46 non-injected DNA. Lane 6 contains DNA from a stage 46 embryo injected with CMV-EGFP-DI-attB plasmid alone. Lane 7 contains DNA from a stage 46 embryo injected with CL-EGFP-DI-attB plasmid alone. Lane 8 contains 10 pg of CMV-DI-EGFP-attB plasmid linearized with BamHI. Markers in kilobase pairs are indicated to the left of the membrane.
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