| Author | Almeida, Vinicius Cotta de | |
| Author | Schonhoff, Susan | |
| Author | Shibata, Tomoyuki | |
| Author | Leiter, Andrew | |
| Author | Snapper, Scott B. | |
| Access date | 2020-05-01T17:11:16Z | |
| Available date | 2020-05-01T17:11:16Z | |
| Document date | 2003 | |
| 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. | pt_BR |
| ISSN | 1088-9051 | pt_BR |
| URI | https://www.arca.fiocruz.br/handle/icict/41058 | |
| Language | eng | pt_BR |
| Publisher | Cold Spring Harbor Laboratory Press | pt_BR |
| Rights | open access | |
| Subject in Portuguese | Novo método | pt_BR |
| Subject in Portuguese | Vetores | pt_BR |
| Subject in Portuguese | Cromossomos artificiais bacterianos | pt_BR |
| Subject in Portuguese | Escherichia coli | pt_BR |
| Subject in Portuguese | Recombinação homóloga | pt_BR |
| Title | A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria | pt_BR |
| Type | Article | |
| DOI | 10.1101/gr.1356503 | |
| 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. | pt_BR |
| 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. | pt_BR |
| Affilliation | New England Medical Center. GRASP Digestive Disease Center. Division of Gastroenterology. Boston, Massachussets, USA / Tufts University School of Medicine, Boston, Massachusetts, USA. | pt_BR |
| 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 . | pt_BR |
| Affilliation | New England Medical Center. GRASP Digestive Disease Center. Division of Gastroenterology. Boston, Massachussets, USA / Tufts University School of Medicine, Boston, Massachusetts, USA. | pt_BR |
| 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 . | pt_BR |
| Subject | New Method | pt_BR |
| Subject | Generating Gene-Targeting Vector | pt_BR |
| Subject | Homologous Recombination | pt_BR |
| Subject | Bacteria | pt_BR |
| Subject | Escherichia coli | pt_BR |
| e-ISSN | 1549-5469 | |
