Identification of effective siRNA sequence for RelB silencing in murine dendritic cells with siRNA cassette / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct small interfering RNA (siRNA) expression cassette targeting murine RelB gene and identify the most effective siRNA sequence against RelB gene in murine bone marrow-derived dendritic cells (DCs).</p><p><b>METHODS</b>Three expression cassettes namely R1/siRNA, R2/siRNA and R3/siRNA targeting the sites 1027, 302 and 1121 of RelB gene, respectively, were constructed by PCR approach and transfected into cultured murine myeloid DCs by catione liposome Advant-Gene. After incubation for 24 hours in a incubator containing 5% CO(2) at 37 degrees C, the DCs were stimulated by lipopolysaccharide (LPS), and RelB gene expression in DCs were then detected by RT-PCR and immunofluorescence.</p><p><b>RESULTS</b>RT-PCR and immunofluorescence assay showed that the expression of RelB gene in DCs transfected with R2/siRNA could not be upregulated by LPS stimulation, but transfection with R1/siRNA or R3/siRNA failed to produce such effect.</p><p><b>CONCLUSION</b>R2/siRNA is an effective sequence for RelB silencing, and can be a useful means to construct new tolerogenic DC, RNAi RelB DC, for clinical immunotolerance induction.</p>