Lentivirus-mediated stable Fas gene silencing in human umbilical cord-derived mesenchymal stem cells / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Lentivirus-mediated RNAi can effectively inhibit Fas gene expression in cultured UC-MSCs.</p>