Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells （DCs） were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time ,pre-electroporation cell condition and serum concentration in electrical buffer, lnverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency （22.10%） could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer. Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.
RNA interference(RNAi),which causes the degradation of any RNA in a sequence specific manner,is a posttranscriptional gene silencing mechanism.Targeting the invariant chain(Ii)in DCs has been used as an approach to enhance antitumor immunity.It is demonstrated in this article that transfection of H-2(K)DCs with siRNA specific for Ii gene can significantly knock down Ii.When exposed to TNF-alpha,immature DCs transfected with Ii siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production.Ii siRNA-treated H-2(K)DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay.Furthermore,Ii siRNA-transfected H-2(K)DCs enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production,and much stronger cytotoxic activity was observed when DCs were co-transfected with Ii siRNA and an endogenous tumor antigen in vitro.Our findings indicate that silencing the Ii gene in DCs with siRNA may offer a potential approach to enhancing antitumor immunotherapy.
Background Genetic modification of dendritic cells （DCs） has been used as an effective approach to enhance anti-tumor immunity. RNA interference （RNAi）, which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA （siRNA） specific for the li gene was transfected into DCs, and the anti-tumor immunity of li-silenced DCs was assessed. Methods The silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96 non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used. Results The li expression of DCs was significantly reduced after li siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with li siRNA plus endogenous tumor antigen （P 〈0.05）. Furthermore tumor growth was greatly inhibited when mice were immunized with DCs transfected with li siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4^= and CD8^＋ T cells were significantly activated in the li siRNA group （P 〈0.05）. Conclusion Silencing of the li gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.
KE ShanCHEN Xue-huaZHU Zheng-gangLI Jian-fangYU Bei-qinGU Qin-longLIU Bing-ya
