Abstract: Objective To construct a lentivirus-mediated vector for RNA interference (RNAi) of Fas and establish a human
umbilical cord-derived mesenchymal stem cells (UC-MSCs) line with stable Fas gene silencing. Methods Four short hairpin
RNA sequences targeting the coding region of human Fas mRNA were designed. The synthesized oligonucleotides were
ligated with the lentivirus vectors harvested from BamHI and EcoRI double digestion of LV3 recombinant vector. The
recombinant lentivirus vectors were transfected into the packaging cells 293T, and the lentivirus titers were determined.
Cultured UC-MSCs were infected with the lentivirus, and real-time PCR and Western blotting were used to detect the
expressions of Fas mRNA and protein in the transfected cells. Results Restriction digestion and DNA sequencing showed that
the lentiviral vectors were successfully constructed, and the titer of lentivirus reached 3 × 108 TU/ml in the packaging cells.
Real-time PCR and Western blot demonstrated significantly suppressed Fas gene expression in UC-MSCs after infection with
the recombinant lentivirus. Conclusion Lentivirus-mediated RNAi can effectively inhibit Fas gene expression in cultured
UC-MSCs.