Construction of conditional knockout targeting vectors using the new recombineering reagents

<p><b>Copyright information:</b></p><p>Taken from "A recombineering based approach for high-throughput conditional knockout targeting vector construction"</p><p></p><p>Nucleic Acids Research 2007;35(8):e64-e64.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885671.</p><p>© 2007 The Author(s)</p> () The genomic structure of a locus with exons 3–5 to be deleted in the cko allele. The cassette flanked by two rare cutter sites, I-SceI and I-CeuI, is targeted to the 5′ side of the intended deletion region. Subsequently, the () cassette is targeted to the 3′ side of the deletion region. The point mutation present in the coding sequence of PL452 and PL451 plasmids (,) was corrected in this cassette which resulted in higher resistance to Kanamycin in and a 2-fold increase in the number of G418-resistant ES colonies. Coloured lines represent the short homology arms in recombineering. () The genomic DNA fragment is retrieved from the BAC to PL611, which has the Amp gene. In a typical cko vector, we choose 4–5-kb genomic DNA as the left homology arm (5′), and 2–3 kb as the right homology arm (3′). The genomic DNA region to be deleted is generally between 1 and 7 kb. () The cassette can conveniently be replaced by a reporter, i.e. , in a simple ligation reaction. The final targeting vector has the reporter flanked by two sites followed by a P site at the 5′ side of the intended deletion region, and a flanked cassette providing positive selection in ES cells. The negative selection marker is added to the vector backbone by recombineering. The vector is linearized with the rare-cutter I-PpoI