Simplified plasmid rescue of host sequences adjacent to integrated proviruses |
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| Institution: | 1. UCA, INRA, UMR PIAF, F-63000 Clermont-Ferrand, France;2. National Institute of Agronomy of Tunisia (INAT), Sylvo-Pastoral Laboratory of Tabarka, Tunisia;3. Institut de l’Olivier, LR: Amélioration et Protection des Ressources Génétiques de l’Olivier-Université de Sfax, Tunisia;4. National Institute of Agronomy of Tunisia (INAT), Crop Improvement Laboratory, INRAT, Tunisia;5. UCA, INRA, UMR 1095 Génétique, Diversité et Ecophysiologie des Céréales, BP 10448, F-63000 Clermont-Ferrand, France;6. CIRAD, UMR AGAP, F-63000 Clermont-Ferrand, France;7. Université d’Orléans, Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, INRA-USC1328, F-45067 Orléans, France;1. Provincial Key Laboratory of Solid State Optoelectronic Devices, Zhejiang Normal University, 321004, China;2. School of Sciences, Zhejiang Sci-Tech University, 310018, China;1. The Molecular Biology of Metabolism Laboratory, The Francis Crick Institute, London, NW1 1AT, United Kingdom;2. Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, CB2 1GA, United Kingdom;3. Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96, Göteborg, Sweden;4. Department of Surgery and Cancer, Division of Computational and Systems Medicine, Imperial College London, London, SW7 2AZ, United Kingdom;5. Department of Genetics, Evolution and Environment, University College London, WC1E 6BT, London, United Kingdom;1. UNESP − Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias-FCAV, Depto. de Biologia Aplicada à Agropecuária (DBAA), 14884-900 Jaboticabal, SP, Brazil;2. UnB − Universidade de Brasília, Instituto de Ciências Biológicas Depto. de Botânica, 70910-900, Brasília, DF, Brazil;3. UFG − Universidade Federal de Goiás, Instituto de Ciências Biológicas, Depto. de Botânica, 74001-970, Goiânia, GO, Brazil |
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| Abstract: | We have previously described a Moloney murine leukemia retroviral (MoMLV) vector useful for the generation of anchored long-range maps of complex mammalian genomes. We now report the development of a modified vector carrying the ColE1 origin of replication and the chloramphenicol-resistance (CmR) gene to facilitate the recovery of genomic sequences adjacent to integrated proviruses. We demonstrate the utility of this new vector for the rescue in plasmid form of a 6-kb human fragment containing portions of an Alu element adjacent to the proviral 3′-LTR from an infected human primary fibroblast clone. We generated a sequence-tagged site (STS) from the derived sequence outside the Alu element, and used a somatic cell hybrid mapping panel to assign this STS to human chromosome 10 by a polymerase chain reaction-based assay. We suggest that this new CmR vector will be useful for recovering sequences of genes interrupted by proviral insertion, to generate directional maps with respect to the inserted provirus using the single cleavage sites within the vector, and to generate site-specific STS for defining and mapping the integration site. |
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