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Analyse des réponses cellulaires induites par l'intégration d'un ADN étranger au sein du génome

Abstract : In numerous situations, cell genome integrity could be in danger. This is the case during gene movements (translocations, mobiles elements), during viral infections that could be sometimes integrative (AAV, HBV, HPV) or during retroviral infections. Indeed, the replication cycle of retroviruses requires a viral genome integration step. In spite of the studies on HIV infections, viral or non viral cancers and retrovirus-based gene therapy, no data are available concerning the cellular modifications induced by such chromosomal disruptions. The aim of work was to identify cellular mechanisms induced by the insertion of a non cellular DNA into the chromosomes. The integration of additional DNA was provoked by HIV-1-based lentiviral vectors. Cellular modifications only due to the integration step were isolated by comparison of integrative and non integrative vectors. Primary human dermal fibroblasts cells were selected for the study. Optimal times post infection and viral quantities were defined using kinetic integration experiments and integrated viral DNA quantifications. The study of cellular modifications induced by the integration of the foreign DNA was applied on the cellular global transcriptome and proteome. In order to perform a transcriptomic analyses, DNA microarray corresponding to the whole human genome, were used. This study revealed a strong transcriptional repression induced by the integration. Moreover, every cellular function are disturbed by the process. Finally, a network based on molecular interactions and biological functions underlined five cellular processes mostly affected by the foreign DNA integration and corresponding to the cell cycle and death, the remodelling and repair of chromatin and the immunity or stress responses. To complete this transcriptomic analyses, a proteomic study was realized. Cellular proteins were separated on 2D gels. Among the nine proteins identified by mass spectrometry, some are linked to the cytoskeleton and other to the cellular stress. Thus, integration of a foreign DNA into the genome provoked cellular perturbations. As additional DNA integration do not only concern retroviruses, data obtained during this study could allow (i) the development of defensive strategies against retroviruses or other diseases implicating the genome integrity and (ii) the evaluation of risks linked to the integration of a therapeutic vector into the genome during gene therapy and gene transfer experiments.
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https://tel.archives-ouvertes.fr/tel-00550487
Contributor : Virginie Gay <>
Submitted on : Tuesday, December 28, 2010 - 2:17:03 PM
Last modification on : Wednesday, October 14, 2020 - 3:44:18 AM
Long-term archiving on: : Tuesday, March 29, 2011 - 2:53:12 AM

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  • HAL Id : tel-00550487, version 1
  • PRODINRA : 246857

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Virginie Gay. Analyse des réponses cellulaires induites par l'intégration d'un ADN étranger au sein du génome. Sciences du Vivant [q-bio]. Université Claude Bernard - Lyon I, 2010. Français. ⟨tel-00550487⟩

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