Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction
| Autor: | O W Merten, Sofia Benkhelifa-Ziyyat, Anne Galy, Sabine Charrier, M Ferrand, J Perea, M Zerbato, A. Viornery, G Précigout, Bucher-Laurent S |
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| Přispěvatelé: | Laboratoire Environnements Sédimentaires - Géosciences Marines (GM/LES), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Institut de l’Élevage, Vectorologie et transfert de gènes (VTG / UMR8121), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes (IMBI), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, INTA - Instituto Nacional de Tecnología Agropecuaria, Généthon, École Pratique des Hautes Études (EPHE), Laboratoire Environnements Sédimentaires (LES), Géosciences Marines (GM), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Institut de l'élevage (IDELE) |
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Genetic enhancement
Virus Integration [SDV]Life Sciences [q-bio] Genetic Vectors Population Antigens CD34 Biology Viral vector Cell Line Hematopoietic Stem Cells CD34+ cell 03 medical and health sciences Transduction (genetics) Transduction 0302 clinical medicine Genetic Transduction Genetic hematopoietic cells Genetics Humans Progenitor cell Antigens education Molecular Biology 030304 developmental biology 0303 health sciences education.field_of_study Q-PCR Stem Cells Lentivirus Gene Transfer Techniques Lentivirus/*genetics CD34/metabolism Cell biology Haematopoiesis CFC Cell culture 030220 oncology & carcinogenesis method [SDV.IMM]Life Sciences [q-bio]/Immunology Molecular Medicine Original Article Stem cell |
| Zdroj: | Gene Ther Gene Ther, 2011, 18 (5), pp.479-87. ⟨10.1038/gt.2010.163⟩ Gene Therapy Gene Therapy, 2011, 18 (5), pp.479-487. ⟨10.1038/gt.2010.163⟩ |
| ISSN: | 0969-7128 1476-5462 |
| DOI: | 10.1038/gt.2010.163⟩ |
| Popis: | International audience; Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies. |
| Databáze: | OpenAIRE |
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