Abstract
The two most common methods used to generate transgenic Xenopus embryos, restriction enzyme-mediated insertion, and I-SceI meganuclease take advantage of relatively common but spatially unpredictable double-stranded breaks in sperm, egg, or early embryo genomes. These methods also tend to insert multimeric copies of the transgene. An alternative is to use bacteriophage- or transposon-derived integrase or recombinase to mediate more site-specific insertion of the transgene. The use of phiC31 integrase requires a defined sequence for insertion and is compatible with insertion of a single copy of the transgene. We describe the protocol we use to facilitate phiC31 integrase transgene insertion including the use of insulator sequences to reduce position effect disruption of transgene activity.
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Acknowledgements
The authors would like to thank Michele Calos for providing the pET11ΦC31 poly(A) plasmid, Gary Felsenfeld and colleagues for providing the HS4 insulator sequences, Paul Krieg for providing the γ-crystallin lens promoter, and Tim Mohun for providing the Nkx2-5 promoter. This work was supported by funding from the NIH (GM069944 and DC007481). Bryan Allen was a student in the Medical Scientist Training Program at the Roy J. and Lucille A Carver College of Medicine, University of Iowa.
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Li, Y.E., Allen, B.G., Weeks, D.L. (2012). Using ΦC31 Integrase to Mediate Insertion of DNA in Xenopus Embryos. In: HOPPLER, S., Vize, P. (eds) Xenopus Protocols. Methods in Molecular Biology, vol 917. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-992-1_13
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DOI: https://doi.org/10.1007/978-1-61779-992-1_13
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