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A NEW METHOD FOR RAPIDLY GENERATING GENE-TARGETING VECTORS BY ENGINEERING BACS THROUGH HOMOLOGOUS RECOMBINATION IN BACTERIA
Almeida, Vinicius Cotta de et al. | Date Issued: 2003
Author
Almeida, Vinicius Cotta de
Schonhoff, Susan
Shibata, Tomoyuki
Leiter, Andrew
Snapper, Scott B.
Affilliation
Massachusetts General Hospital. Gastrointestinal Unit. Center for the Study of Inflammatory Bowel Disease. Boston, MA, USA / Harvard Medical School. Department of Medicine. Boston, MA, USA / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Ultraestrutura e Biologia Celular. Rio de Janeiro, RJ, Brasil.
New England Medical Center. GRASP Digestive Disease Center. Division of Gastroenterology. Boston, Massachussets, USA / Tufts University School of Medicine, Boston, Massachusetts, USA.
Massachusetts General Hospital. Gastrointestinal Unit. Center for the Study of Inflammatory Bowel Disease. Boston, MA, USA / Harvard Medical School. Department of Medicine. Boston, MA, USA .
New England Medical Center. GRASP Digestive Disease Center. Division of Gastroenterology. Boston, Massachussets, USA / Tufts University School of Medicine, Boston, Massachusetts, USA.
Massachusetts General Hospital. Gastrointestinal Unit. Center for the Study of Inflammatory Bowel Disease. Boston, MA, USA / Harvard Medical School. Department of Medicine. Boston, MA, USA .
Abstract
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.
Keywords in Portuguese
Novo método
Vetores
Cromossomos artificiais bacterianos
Escherichia coli
Recombinação homóloga
 
Keywords
New Method
Generating Gene-Targeting Vector
Homologous Recombination
Bacteria
Escherichia coli
 
Publisher
Cold Spring Harbor Laboratory Press
Citation
ALMEIDA, Vinicius Cotta de et al. A New Method for Rapidly Generating Gene-Targeting Vectors by Engineering BACs Through Homologous Recombination in Bacteria. Genome Research, v. 13, p. 2190-2194, 2003.
DOI
10.1101/gr.1356503
ISSN
1088-9051