title: [Lentivirus-mediated stable Fas gene silencing in human umbilical cord-derived mesenchymal stem cells].
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.
  
  
  CONCLUSIONS
  Lentivirus-mediated RNAi can effectively inhibit Fas gene expression in cultured UC-MSCs.
authors.affiliation: Clinical Laboratory and Diagnostic Center, Bengbu Medical College, Bengbu 233030, China.E-mail: wanglu14712@163.com.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhai
authors.forename: Xiangrong
authors.initials: X
authors.lastname: Yang
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Yuyun
authors.initials: Y
authors.lastname: Li
mesh_terms: D002478:Cells, Cultured; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D059630:Mesenchymal Stem Cells; D034622:RNA Interference; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D014470:Umbilical Cord / Q000166:cytology; D019014:fas Receptor / Q000235:genetics*
chemical_list: C491901:FAS protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019014:fas Receptor