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. Inverted 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.
