RESUMO
BACKGROUND: Inhibition of viral genes through siRNA seems to be promising for treatment of complicated viral infections like human immunodeficiency virus (HIV-1). HIV-1 Tat (Trans Activator of Transcription) and Nef (Negative regulatory Factor) proteins are very interesting targets for designing siRNAs. METHODS: The effectiveness of suppressing Tat and Nef was investigated using three specific siTATs and three siNEFs. They were used to transfect the developed stable and infected Human Embryonic Kidney cells (HEK293) as an ex-vivo model. Both stable and virus infected HEK293 cells were transfected with each siTAT and siNEF. The inhibitory effect was evaluated using qRT-PCR, western blot analysis, and HIV P24 ELISA. RESULTS: siTAT-100, siTAT-162, and siNEF-136 and at a concentration of 100 nM/mL showed the most inhibitory effect on their target genes. CONCLUSIONS: Utilization of more developed molecular inhibition strategies such as RNAi or even a combination of different molecular approaches could be promising to overcome emerging HIV escape mutants.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/genética , Interferência de RNA , Replicação Viral/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Células HEK293 , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodosRESUMO
OBJECTIVES: In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identified. BACKGROUND: Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like Turbofect. Transfection efficiency could be affected by the nature of target cell line and vector. METHODS: Transfection efficiency and cytotoxicity of each reagent was identified by using flow cytometry and XTT assay, respectively. RESULTS: Lipofectamine 3000 was more efficient in transfecting pCDH, while Turbofect was more efficient in separate transfection of CHO-K1 and HEK293 with pEGFP-N1. Lipofectamine 3000 could be cytotoxic in transfecting H9T-cells with pCDH. Also, H9T-cells were not sufficiently transfected with each plasmid vector by using each Lipofectamine 3000 and Turbofect. Turbofect had less cytotoxicity effect on all three cell lines than Lipofectamine 3000.Transfection of suspended cells like H9T-cells by using Lipofectamine 3000 and Turbofect would not result in sufficient transfection. CONCLUSION: Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. 1, Fig. 2, Ref. 26).
