Effects of RNA interference on CD80 and CD86 expression in bone marrow-derived murine dendritic cells.
To investigate whether RNA interference (RNAi) induced by small interfering RNA (siRNA) could suppress CD80 and CD86 expression in bone marrow-derived murine dendritic cells (DC). The bone marrow-derived DC of mice were separated and cultured in vitro, chemically synthesized siRNA were then transferred into the cells by LipofectAMINE 2000, and the siRNA transfection efficacy was assessed by both fluorescence microscope and flow cytometry. The mRNA expression and protein synthesis were analysed by real-time RT-PCR and flow cytometry. The cell viability of transfected DC was determined by annexin V and propidium iodine staining. Transfection of bone marrow-derived murine DC with a non-silencing FITC-labelled control siRNA demonstrated a high (71.86%) transfection efficiency without affecting cellular viability. CD80-1 siRNA was the most effective siRNA to block CD80 expression in three candidates. Similarly, CD86-3 siRNA was extraordinarily effective in repressing the expression of CD86. Cotransfection of siRNA specific to CD80 and CD86 can enhance gene silencing that is not affected by DC activation-inducing signals. CD80 and CD86 siRNA suppressed the expression of CD80 and CD86 to 31.05 +/- 2.41% and 25.43 +/- 0.85%, respectively, of the level in untreated cells (P < 0.05). siRNA is capable of triggering RNAi in bone marrow-derived DC; it can specifically and effectively knock down CD80 and CD86 gene expression. This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for allograft rejection.
Gu
X
X
Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, China.
Xiang
J
J
Yao
Y
Y
Chen
Z
Z
D000818:Animals; D018122:B7-1 Antigen / Q000235:genetics*; D051940:B7-2 Antigen / Q000235:genetics*; D001854:Bone Marrow Cells / Q000737:chemistry / Q000187:drug effects* / Q000276:immunology; D003713:Dendritic Cells / Q000737:chemistry / Q000187:drug effects* / Q000276:immunology; D015870:Gene Expression / Q000187:drug effects; D051379:Mice; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D014162:Transfection
D018122:B7-1 Antigen; D051940:B7-2 Antigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering