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Updated: Aug 9, 2026
Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation
Published on: July 8, 2011
[Identification of effective siRNA sequence for RelB silencing in murine dendritic cells with siRNA cassette]
Objective:
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).
Methods:
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.
Results:
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.
Conclusion:
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.
