title: [Construction and identification of SPARC shRNA lentiviral vector and its expression in human SKM-1 cells].
abstract: OBJECTIVE
  To construct a lentiviral vector expressing small-hairpin RNA(shRNA) targeting SPARC gene and investigate its silenced effect on SPARC in human myelodysplastic syndromes(MDS) cell line SKM-1.
  
  
  METHODS
  The targeting sequence of SPARC gene which can be effectively silenced in RNA interference was confirmed in our previous study. The designed and synthesized single-stranded primers were annealed to double-stranded oligo sequences and subcloned into linear pGCSIL-GFP lentiviral plasmid digested by enzyme Age I and EcoR I to produce GC-shSPARC lentiviraL vector. After being identified by PCR and sequencing,  plasmids GC-shSPARC with pHelper 1.0 and pHelper 2.0 were cotransfected into 293T cells to package lentiviral particles. The recombinant lentiviral vector was transfected into human SKM-1 cells,  transfection efficiency was evaluated with expression of green fluorescent protein(GFP) determined by fluorescent microscope. Expression of SPARC in SKM-1 cells was detected using RT-PCR and Western blotting.
  
  
  RESULTS
  A recombinant lentiviral vector,  GC-shSPARC,  expressing shRNAs targeting SPARC gene was constructed and confirmed by DNA sequencing. The recombinant lentivirus was harvested from 293T cells with a viral titer of 1×10(9); TU/mL. GFP was observed in the 70% of SKM-1 cells after transfection. Expression of SPARC mRNA and protein was significantly reduced in the GC-shSPARC transfected group than that in the control  group (P<0.05).
  
  
  CONCLUSIONS
  The lentivirus RNAi vector targeting SPARC has been successfully constructed,  and can effectively inhibit the expression of SPARC in SKM-1 cell line,  which shed light on the foundation for researching the inhibition of SPARC siRNA target against human MDS cells proliferation,  induction apoptosis and gene therapy.
authors.affiliation: Department of Hematology, the First Affiliated Hospital, Chongqing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Luo
authors.forename: Xing-wei
authors.initials: XW
authors.lastname: Ye
authors.forename: Wen
authors.initials: W
authors.lastname: Jiang
authors.forename: Hong-jing
authors.initials: HJ
authors.lastname: Zhou
authors.forename: Qing
authors.initials: Q
authors.lastname: Xiao
authors.forename: Ze-song
authors.initials: ZS
authors.lastname: Yang
authors.forename: Lin
authors.initials: L
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Wang
mesh_terms: D002460:Cell Line; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009190:Myelodysplastic Syndromes / Q000235:genetics / Q000628:therapy*; D015676:Osteonectin / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D015676:Osteonectin; D034741:RNA, Small Interfering