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.
BACKGROUND:A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations,which promote tumor progression. Epigenetic abnormalities include changes in DNA methylation and in the modification of histones.This study aimed to assess the status of methylation in the CpG island(CGI)of the tumor necrosis factor receptor superfamily member 10c(TNFRSF10C) with combined bisulfite restriction analysis(COBRA)and to evaluate its role in the progression of pancreatic cancer(PC). METHODS:The methylation status of four PC cell lines was assessed using COBRA and/or bisulfite genomic sequencing (BGS).Changes in methylation and TNFRSF10C expression in PC cell lines before and after treatment with 5-aza-2’-deoxycytidine(5-aza-dC)and/or trichostatin A(TSA)were assessed by BGS and real-time RT-PCR.Apoptosis in the four cell lines was tested by flow cytometry(FCM)and TUNEL assay. RESULTS:The methylation status of the TNFRSF10C promoter was assessed in PC cells(BxPC-3:68.84±8.71%;CFPAC-1:0; PANC-1:96.77±4.57%;SW1990:54.97±7.33%)with the COBRA assay,which was confirmed by the results of BGS.After treatment with 5-aza-dC and/or TSA,apoptosis was induced in PC cells to different degrees,and the levels of TNFRSF10C transcriptional expression in the PC cell lines(except CFPAC-1) increased markedly after 5-aza-dC treatment. CONCLUSIONS:A high frequency of CGI methylation in the TNFRSF10C promoter results in inactivation of the gene and enhancement of tumor growth in most PC cell lines(except CFPAC-1).Inactivation of TNFRSF10C by CGI hypermethylation can play an important role in PC progression and be potentially useful as a diagnostic marker and a new therapeutic approach for PC.
Hui-Hua Cai,Yue-Ming Sun,Yi Miao,Wen-Tao Gao,Quan Peng,Jie Yao and Han-Lin Zhao Department of General Surgery,First Affiliated Hospital, Nanjing Medical University,Nanjing 210029,China
