[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.


CONCLUSIONS
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.
Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. nflab@163.com
Lei
L
Zheng
Jie
J
Bao
Qian
Q
Wang
Hong-ling
HL
Yang
Yu-rong
YR
Qiu
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051841:Transcription Factor RelB / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D008070:Lipopolysaccharides; C497593:RELB protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051841:Transcription Factor RelB