Development of new EBV-based vectors for stable expression of small interfering RNA to mimick human syndromes: application to NER gene silencing.
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.
Biard
Denis S F
DS
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
Despras
Emmanuelle
E
Sarasin
Alain
A
Angulo
Jaime F
JF
D015139:Blotting, Southern; D015153:Blotting, Western; D002453:Cell Cycle / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics; D005434:Flow Cytometry; D020868:Gene Silencing; D005822:Genetic Vectors / Q000502:physiology*; D006367:HeLa Cells / Q000502:physiology / Q000528:radiation effects; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins; D014410:Tumor Stem Cell Assay; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; C489487:KIN protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C086984:XPC protein, human