A recombineering based approach for high-throughput conditional knockout targeting vector construction

Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies

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

Increasing the recombineering efficiency by inoculating different amounts of cells in 96-well plates

<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> cells harbouring BACs are growing differently due to the nature and the sizes of genomic DNA inserts. Efficient recombineering and transformation require cells growing in log phase. To increase the likelihood that cells of a particular BAC reach the optimal growth condition prior to heat induction and recombineering, four 96-well plates were used for the 2 h culture. Each plate had different amounts of the overnight culture. Cells from the four plates were combined after heat induction and were transformed with PCR products