Construction and packaging of the recombination lentivirus vector with RNA interference gene targeting human Fas

Objective:To construct the recombination lentivirus vector with short hairpin RNA gene targeting human factor associated suicide(Fas), and silence the Fas gene expression in umbilical cord mesenchymal stem cells.Methods:According to Fas gene mRNA sequence, four groups of the short hairpin RNA gene sequence targeting Fas were designed, synthesised and annealed to form double-stranded DNA fragments, which was inserted the LV3 vector digested by BamHⅠ and EcoRⅠ enzyme, then it was transfected into the competent cells.The positive clones were identified by PCR, and analyzed by sequencing.The packaging and titer of lentivirus were determined after transfecting 293T cells.Results:Lentiviral vector was constructed successfully by the identification of restriction enzyme digestion and gene sequencing.The lentivirus vector was transfected into 293 T cells using the fluorescence microscope observation, the infection efficiency of which was more than 90%.The high titer of Lentiviral vector was obtained, which was 3×10⁸ TU/ml.Conclusions:The recombination lentivirus vector with interference gene targeting human Fas is successfully constructed, which can provide the basis of transfecting umbilical cord mesenchymal stem cells.