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Methods

A New Method for Rapidly Generating Gene-Targeting Vectors by Engineering BACs Through Homologous Recombination in Bacteria

¹ Gastrointestinal Unit and the Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, Massachusetts 02114, USA , ² Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA , ³ Department of Ultrastructure and Cell Biology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, 21045-900, Brazil , ⁴ Division of Gastroenterology, GRASP Digestive Disease Center, New England Medical Center/Tufts University School of Medicine, Boston, Massachusetts 02111, USA

Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1356503.

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