Identification of effective siRNA sequence for RelB silencing in murine dendritic cells with siRNA cassette.
- Author:
Lei ZHENG
1
;
Jie BAO
;
Qian WANG
;
Hong-ling YANG
;
Yu-rong QIU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Bone Marrow Cells; cytology; Cells, Cultured; Dendritic Cells; cytology; drug effects; metabolism; Female; Fluorescent Antibody Technique; Gene Expression; drug effects; Lipopolysaccharides; pharmacology; Male; Mice; Mice, Inbred C57BL; RNA Interference; RNA, Messenger; biosynthesis; genetics; metabolism; RNA, Small Interfering; genetics; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor RelB; biosynthesis; genetics; Transfection
- From: Journal of Southern Medical University 2006;26(3):301-304
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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).
METHODSThree 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.
RESULTSRT-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.
CONCLUSIONR2/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.
