International Mouse Knockout Consortium, Collins, F.S., Rossant, J. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Establishment in culture of pluripotential cells from mouse embryos. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Site-directed, mutagenesis by gene targeting in mouse embryo-derived stem cells. This protocol needs ∼2 weeks to construct a targeting vector, and several vectors can be easily handled simultaneously using common laboratory setup. To demonstrate the usefulness of this protocol, we report targeted disruptions of members of the cadherin gene family, focusing on those that have not been previously studied at the molecular genetic level. Finally, through a simple in vitro Gateway recombination, the modified genomic fragment is switched into a vector that contains negative selection cassettes, as well as unique sites for linearization. ![]() ![]() Second, the vector is modified by recombineering to include a positive selection gene neo, from a variety of modular reagents. First step is the use of Red-pathway-mediated recombination (recombineering) to capture a genomic fragment into a Gateway-compatible vector. This procedure combines various highly efficient recombination-based cloning methods in bacteria, consisting of three steps. We describe here a streamlined procedure for targeting vector construction, which often is a limiting factor for gene targeting (knockout) technology.
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