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Bacteriophage ϕC31 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 ϕC31 integrase approach to make transgenic Xenopus laevis embryos.
The frog, Xenopus laevis, has a long history of use for studies in embryonic development. Previously described Xenopus integration techniques often insert multiple copies of a transgene at random sites in the embryos genome (1–4). Though valuable for many experimental designs, these approaches are problematic for researchers who desire transgene expression to approximate endogenous gene expression levels. Rather, a site-directed integration approach that incorporates a regulated single copy of a transgene into the host genome would more closely match endogenous gene expression. The ϕC31 integrase approach is one way to accomplish this aim.
Bacteriophage ϕC31 encodes an integrase enzyme that inserts the phage genome into the genome of various Streptomyces bacteria (5, 6). The integrase protein recognizes a 39-bp-long phage attachment site (attP) in its own genome and a 34-bp-long bacterial attachment site (attB) in the bacterium’s genome and then catalyzes an integration event (7, 8). The integrase enzyme does not require perfect sequence fidelity to recognize the attP site (7). Non-perfect attP sites, or pseudo-attP sites, may have as low as 24% sequence homology to endogenous attP sites and still allow recombination (7) although the recombination efficiency may be decreased.
Many groups have shown that integrase can insert plasmid DNA sequences that contain an attB site into pseudo-attP sites found in a variety of organisms. Utilizing this approach, transgenes have been inserted into the genomes of plant cells (9), mammalian cells (7,10–18), and Drosophila embryos (19). We used the pseudo-attP sites in the Xenopus genome and an attB site containing reporter plasmid to make transgenic Xenopus embryos (20) (Fig. 9.1). Surprisingly, the reporter genes are expressed in some expected tissues but not in others. We recognized this as chromatin position effect and flanked the reporter gene with HS4 insulators. HS4 insulators stop the spread of chromatin silencing and also prevent distant enhancers from acting on a promoter (21, 22). After making transgenic embryos with the new insulated reporter plasmid, we found that the transgenes expressed as expected (20) (Fig. 9.2). The techniques used to generate and recognize ϕC31 integrase mediated transgenic Xenopus embryos are described below.
We would like to thank Professor Michele Calos for providing the pET11phiC31poly(A) plasmid, Professor Gary Felsenfeld for providing the HS4 insulator sequences, and Paul Kreig for providing the gamma crystallin lens promoter. This work was supported by funding from the NIH (GM069944 and DC007481). Bryan Allen is a student in the Medical Scientist Training Program at the Roy J. and Lucille A Carver College of Medicine, University of Iowa.
1MTA agreements are required from Dr. Gary Felsenfeld (NIH) to use the HS4 insulator sequences and Dr. Daniel Weeks (Iowa) for the CMV-EGFP-DI-attB and CL-EGFP-DI-attB plasmids. An MTA is also required to obtain the plasmid pET11phiC31poly(A) from Dr. Michele Calos (Stanford).
2The amount of fluorescence produced from a single-copy transgene (ϕC31 integrase approach) may be significantly less than an embryo containing multiple copies of a transgene generated from the restriction enzyme mediated insertion (1, 2) approach or the meganuclease (3, 4) approach.