[Construction and identification of SPARC shRNA lentiviral vector and its expression in human SKM-1 cells]

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.

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.

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).

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.