Construction and identification of lentiviral vector of RNA interference of GFβ1 gene

Objective: To construct a lentiviral vector of RNA interference(RNAi) of TGF-β1 gene. Methods: Three target sequences were selected according to TGFβ1 mRNA sequence firstly,then three pairs of oligonucleotide sequences according to these target sequences and one pair of negative control oligonucleotide sequence were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pRNA-U6/Lenti plasmid. After identified by restriction enzyme digestion and sequencing ,these plasmids were cotransfected along with ViraPowerTM Lentiviral Packaging Mix into 293FT cells for the package of lentiviral particles. Then the lentiviral vector particles were transfected into Hela cells and TGF-β1 expression in the transfected cells was assayed by Real-Time PCR and ELISA. Results: It was confirmed by digestion and sequencing that lentiviral vectors had the correct structure and could express high titer of virus. After transfected into Hela cells,TGF-β1 expression was decreased significantly by two of these lentiviral vectors at both the protein and mRNA level and the first vector had the best interefence efficiency. Conclusion: The lentivirus RNAi vector of TGF-β1 was constructed successfully.