RESUMEN
In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.
Asunto(s)
Humanos , Línea Celular Tumoral , Dependovirus , Genética , Metabolismo , Doxiciclina , Farmacología , Ganciclovir , Farmacología , Genes Transgénicos Suicidas , Genética , Terapia Genética , Vectores Genéticos , Genética , Simplexvirus , Genética , Timidina Quinasa , Genética , TransfecciónRESUMEN
<p><b>BACKGROUND</b>RevTet-On gene expression system was used to deliver the suicide gene tk to human breast cancer cell line MCF-7 and control the tk gene expression level. The animal model of human breast cancer on severe combined immune deficiency (SCID) mice was set up to explore the suicide gene therapy by the regulation of Tet-On.</p><p><b>METHODS</b>Herpes simplex virus-thymidine kinase (HSVtk) gene was inserted into the plasmid pRevTRE and the recombinant retroviral vector pRevTRE/HSVtk was constructed. Using modified calcium phosphate co-precipitation method, two transfections, pRevTRE/HSVtk and pRevTet-On were performed for MCF-7 cell line and selected by hygromycin B and G418. MCF-7 cell line that stably expressed Tet-regulated tk gene was established. HSVtk gene expression in the MCF/TRE/tk/Tet-On cell line was under the control of Doxycycline (Dox). Cell viability was also determined by MTT assay, whereas HSVtk gene expression was analyzed by reverse transcription-PCR (RT-PCR).</p><p><b>RESULTS</b>MCF/TRE/tk/Tet-On cell survival rate was decreased from 100% to less than 20% when ganciclovir (GCV) concentration was increased from 0 to 1000 microg/ml at 1 microg/ml of Dox after 72 hours of GCV administration. At 1 microg/ml of GCV concentration, the cell numbers decreased from 7 x 10(4) cells/ml to 2 x 10(4) cells/ml when Dox concentration was increased from 0 to 1500 ng/ml after 72 hours culture. In addition, bystander effects were generated in vitro when 10% - 25% of transduced MCF-7 cells were mixed in untransduced MCF-7 cells. On the other hand, the human breast cancer models in SCID mice were set up. The tk gene was expressed with the regulated character after MCF/TRE/tk/Tet-On cells were implanted into the female SCID mice 7 days after Dox induction followed by intraperitoneally administration of GCV for 23 days. Subcutaneous tumors in SCID mice that were implanted with MCF/TRE/tk/Tet-On cells shrank remarkably after Dox and GCV administration as compared with the control.</p><p><b>CONCLUSION</b>The human breast tumor cells (MCF-7) expressing HSVtk gene can be eradicated by administration of GCV and induced with tetracycline or its derivative Dox in vitro and in vivo.</p>
Asunto(s)
Animales , Humanos , Ratones , Neoplasias de la Mama , Terapéutica , Efecto Espectador , Línea Celular Tumoral , Supervivencia Celular , Doxiciclina , Farmacología , Ganciclovir , Farmacología , Genes Transgénicos Suicidas , Terapia Genética , Métodos , Vectores Genéticos , Herpesviridae , Genética , Ratones SCID , Retroviridae , Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina Quinasa , Genética , TransfecciónRESUMEN
<p><b>OBJECTIVE</b>To explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus.</p><p><b>METHODS</b>The recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus, which was produced from cloned PA317 cells screened by hygromycin B after "micro-pingpong" technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration.</p><p><b>RESULTS</b>The recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus.</p><p><b>CONCLUSION</b>High titer of retroviruses could be obtained in the culture medium of PA317 cell line through "micro-pingpong" technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.</p>