| Development of new EBV-based vectors for stable expression of small interfering RNA to mimick human syndromes: application to NER gene silencing. | |
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MedLine Citation:
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PMID: 16179499 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPA(KD) and XPC(KD) (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced (HSA)KIN17. Several clones displaying undetectable protein levels of XPA, XPC, or (HSA)kin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, (HSA)KIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPA(KD) and XPC(KD) HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPC(KD) HeLa clones were more sensitive to UVC than their XPA(KD) or KIN17(KD) counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPA(KD) cells recovered a normal UVC sensitivity, XPC(KD) cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, (HSA)kin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles. |
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Authors:
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Denis S F Biard; Emmanuelle Despras; Alain Sarasin; Jaime F Angulo |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Molecular cancer research : MCR Volume: 3 ISSN: 1541-7786 ISO Abbreviation: Mol. Cancer Res. Publication Date: 2005 Sep |
Date Detail:
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Created Date: 2005-09-23 Completed Date: 2005-11-10 Revised Date: 2007-11-15 |
Medline Journal Info:
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Nlm Unique ID: 101150042 Medline TA: Mol Cancer Res Country: United States |
Other Details:
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Languages: eng Pagination: 519-29 Citation Subset: IM |
Affiliation:
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Laboratoire de Génétique de la Radiosensibilité, Commissariat à l'Energie Atomique, Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, BP 6, 92265 Fontenay aux Roses, France. denis.biard@cea.fr |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Blotting, Southern Blotting, Western Cell Cycle / radiation effects Cell Survival / radiation effects DNA Repair* DNA-Binding Proteins / genetics Flow Cytometry Gene Silencing* Genetic Vectors / physiology* Hela Cells / physiology, radiation effects Herpesvirus 4, Human / genetics* Humans Nuclear Proteins / genetics RNA, Small Interfering / genetics* RNA-Binding Proteins Tumor Stem Cell Assay Ultraviolet Rays* Xeroderma Pigmentosum Group A Protein |
| Chemical | |
Reg. No./Substance:
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0/DNA-Binding Proteins; 0/KIN protein, human; 0/Nuclear Proteins; 0/RNA, Small Interfering; 0/RNA-Binding Proteins; 0/XPA protein, human; 0/Xeroderma Pigmentosum Group A Protein; 156533-34-5/XPC protein, human |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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