RESUMEN
Objective To establish a stable transfection cell line of iRhom2 and its mutant through recombinant lentivirus infection.Methods The full-length gene of iRhom2 and its mutant were cloned into the lentivirus vector Lenti-OE-Flag, and got recombinant lentiviral vector of Lenti-OE-iRhom2 and Lenti-OE-iRhom2mut.The constructed recombi-nant lentivirus vectors were transfected into HEK-293T packaging cells to obtain the recombinant virus.Vero cells were in-fected with recombinant virus.The stable expressing cell lines were obtained by pressure screening with puromycin. Results The recombinant lentivirus vectors were constructed and the recombinant virus was obtained.The stable express-ing cell lines were obtained using virus infection and the protein expression was testified with Western blotting.Conclu-sions Stable iRhom2-expressing Vero cell line and its mutant are achieved by recombinant lentivirus infection.It paves the way for future study on biological functions and mechanism of iRhom2.
RESUMEN
A human embryonic kidney cell line 293 is widely used for adenovirus production and propagation. With this cell line, however, replication-competent virus (RCV) is frequently generated, especially during large-scale production and successive propagation because 293 cells contain not only E1 gene but also non-E1 adenovirus gene. Homologous recombination between non-E1 region of 293 genomic DNA and its homologous region in the recombinant adenoviral vector generate RCV. To overcome this problem, we developed a new packaging cell line, Hela-E1, which contains minimum E1 region and from which non-E1 adenoviral region that is homologous with recombinant adenovirus vector was excluded. No RCV was detected during adenovirus propagation in Hela-E1 compared to in 293. In addition, adenovirus-p53 produced in HeLa-E1 was able to overexpress p53 protein when introduced into an ovarian cancer cell line, SKOV3. These results may have a significant impact on the development of packaging cell lines for replication-deficient adenovirus production.
Asunto(s)
Humanos , Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Línea Celular , Genes Virales , Genes p53 , Vectores Genéticos , Células HeLa , Proteína p53 Supresora de Tumor/genética , Recombinación Genética , Células Tumorales Cultivadas , Cultivo de Virus , Replicación ViralRESUMEN
In order to use retroviral-mediated gene transfer technology in clinical application, retroviral vector must be of high titer and free of detectable replication-competent retroviruses (RCR). The aim of this study was to optimize methods of defective retroviral vector production. Study was conducted using a LXSN vector inserted with human tumor necrosis factor-? gene and an amphotropic retrovirus packaging cell line-PA317. The results indicated that viral titer was influenced by volume of medium and concentration of fetal calf serum. Inactivation of retroviral vector was greater at 37癈 than at 32癈. In experiment of transfection of PA317 and transduction of 3T3, integration of retroviral vector into genome of packaging cells and target cells, and free of RCR were detected by polymerase chain reaction analysis. Viral vector with high titer and free of RCR is able to use in clinical trial