Abstract
In recent years, methods to address the simplification of targeting vector (TV) construction have been developed and validated. Based on in vivo recombination in Escherichia coli, these protocols have reduced dependence on restriction endonucleases, allowing the fabrication of complex TV constructs with relative ease. Using a methodology based on phage-plasmid recombination, we have developed a comprehensive TV construction protocol dubbed Orpheus recombination (ORE). The ORE system addresses all necessary requirements for TV construction; from the isolation of genespecific regions of homology to the deposition of selection/disruption cassettes. ORE makes use of a small recombination plasmid, which bears positive and negative selection markers and a cloned homologous “probe” region. This probe plasmid may be introduced into and excised from phage-borne murine genomic clones by two rounds of single crossover recombination. In this way, desired clones can be specifically isolated from a heterogeneous library of phage. Furthermore, if the probe region contains a designed mutation, it may be deposited seamlessly into the genomic clone. The complete removal of operational sequences allows unlimited repetition of the procedure to customize and finalize TVs within a few weeks. Successful gene-specific clone isolation, point mutations, large deletions, cassette insertions, and finally coincident clone isolation and mutagenesis have all been demonstrated with this method.
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Notes
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Why Orpheus? Syrinx (the nymph pursued by the Pan, who changed her shape to that of reeds to escape the God) and Charon (the spectral figure who ferries the dead across the river Styx), two well-known bacteriophage-λ cloning vectors, are the parents of the λTK phage vectors used in our recombination system. In keeping with this naming tradition, we have dubbed the system “Orpheus.” Although Orpheus was the only mortal to enter and depart from the underworld alive, he left a part of himself behind, a suitable parallel to the integrative and excisive recombination used to deposit designed modifications in phage-born clones.
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© 2008 Humana Press Inc., Totowa, NJ
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Woltjen, K., Ito, K., Tsuzuki, T., Rancourt, D.E. (2008). Orpheus Recombination. In: Davis, G.D., Kayser, K.J. (eds) Chromosomal Mutagenesis. Methods in Molecular Biology, vol 435. Humana Press. https://doi.org/10.1007/978-1-59745-232-8_6
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DOI: https://doi.org/10.1007/978-1-59745-232-8_6
Publisher Name: Humana Press
Print ISBN: 978-1-58829-899-7
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