[The development of vector constructions for respiratory syncitial virus (RSV) P-gene silencing]

Interfering RNA (RNAi) is a powerful tool to silence gene expression on the level of mRNA. To knock-down gene expression by using RNAi two major methods of mRNA silencing exist. First method utilizes siRNA (small interfering RNA), a readily processed dsRNA, that enters RISC complex and destroy target mRNA after transfection into the cells. The second method based on the construction of plasmid DNA that expresses shRNA (short harpin RNA) from U6 or CMV promoter. shRNA gets processed by Drosha and Dicer RNAses inside the cell before it translocates to the cell cytoplasm and affects the level of target RNA. In this study we modified lentiviral vector pGIPZ expressing tFP-IRES-Puro-shRNA(mir30) cassette by introducing BamH I restriction site downstream of this cassette. This modification makes possible to clone specific shRNA sequences in pGIPZ vector using XhoI/BamHI restriction sites instead of the original recombination. Three shRNAs against phosphoprotein P of respiratory sinthitial virus (RSV) and shRNA against human CD43 as a control were generated and cloned into modified so-called pCIPD vector. Monkey kidney cells MA-104 were stably transduced with four shRNA constructs. In conclusion, the generated lentiviral vector pCIPD can be successfully used for efficient gene silencing and virus replication in a broad variety of cells.