RESUMEN
Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos/metabolismo , Elementos Aisladores , Factores de Transcripción NFI/metabolismo , Animales , Sitios de Unión , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Factor de Unión a CCCTC , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Virus de la Leucemia Murina de Friend/genética , Virus de la Leucemia Murina de Friend/metabolismo , Silenciador del Gen , Vectores Genéticos/genética , Células HeLa , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFI/genética , Plásmidos/genética , Plásmidos/metabolismo , Proto-Oncogenes/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección , Transgenes , Integración ViralRESUMEN
X-irradiation of C57BL/6 mice induces thymic lymphosarcomas which sometimes contain retroviruses which upon injection into normal mice mimic the effect of the irradiation. We examined whether specific antigenicities, viral or cellular, were expressed by tumour cells that could be recognized by antibodies from the irradiated animals. We developed monoclonal antibodies (MAbs) using splenocytes of the diseased animal. The reactivity of such MAbs towards thymoma cell lines established in vitro was investigated by means of an ELISA. At least 10 antibody specificities were detected on the 13 tumours investigated, allowing separation of the MAbs into three classes: those recognizing the autologous tumour, heterologous tumours as well as normal thymic tissue, those specific for the autologous tumour, and those specific for one tumour, but not ones of autologous origin. The last two classes corresponded to specific tumour-associated antigens. Our panel of MAbs defined each tumour by the particular pattern of antigens harboured. It is striking that most of the antigens were present in the normal thymus and that only two tumours had additional antigenicities. Additionally, quantitative variations were observed in the levels of expression of these antigens.
Asunto(s)
Antígenos de Neoplasias/análisis , Neoplasias Inducidas por Radiación/inmunología , Timoma/inmunología , Neoplasias del Timo/inmunología , Animales , Anticuerpos Monoclonales , Línea Celular , Ratones , Ratones Endogámicos C57BL , Neoplasias Inducidas por Radiación/microbiología , Retroviridae/aislamiento & purificación , Timoma/microbiología , Timo/inmunología , Neoplasias del Timo/microbiologíaRESUMEN
The role of retroviruses in murine radioleukemogenesis was reinvestigated using a protocol associating the injection of a non-pathogenic retrovirus (T1223/B virus) and a subleukemogenic dose of X-radiation (2 X 1.75 Gy). Using the Southern blotting technique we studied MuLV proviral organization and RNA expression in thymic lymphomas induced by the combined effect of virus and irradiation or irradiation alone. A recombinant provirus was detected in the chromosomal DNA of every tumor induced by associating virus and radiation whereas it was unconstantly found in radio-induced tumors. In every instance, the provirus was not integrated at a common site. No relationship was observed between viral RNA expression and tumor induction. Trisomy 15 was observed in all metaphases irrespective of the protocol of tumor induction. The G-banding technique revealed an extra-band in several thymic lymphomas induced by irradiation and T1223/B virus injection.