(A); Western blot analysis shows

a decrease in total DNMT1 in 5 and 10 nmol of DNMT1 siRNA. … Discussion MDA-MB-468 breast cancer cells are hemizygous for a mutated p53 gene, containing a single point mutation and overexpressing a transcribtionaly active mutant p53 protein. These cells are the ER- negative cells with a hypermethylated ER promoter.17 This cell line is a good choice for Sorafenib studying the epigenetic events on ER promoter. DNA methyl transferase Inhibitors,research,lifescience,medical 1 gene has an important role in silencing ER promoter as it has been recently demonstrated that RNAi-mediated DNMT1 knockdown restored the expression of ER gene in this cell line.15 In addition, the ability of genetically-modified MDA-MB-468 breast cancer cell line with a high efficiency provides a new tool for understanding protein-protein interactions in this cell line. For example, through down-regulation of DNMT1, the Inhibitors,research,lifescience,medical binding

capacity of proteins in repression complex that regulates the expression of ER promoter to this hypermethylated promoter Inhibitors,research,lifescience,medical will be studied in future researches.5,17 In addition, transient gene silencing is very useful in studying gene function.5 Electroporation is a promising method for siRNA delivery to cells because the site of action of these molecules is the cytoplasm, where they bind and degrade messenger RNA. Therefore, transport into the nucleus, where transcription occurs, is not necessary. Previous siRNA investigations have applied lipofectamin or cationic lipid formulation to transfer many cells. There are many reports for using electroporation to transfect stem cells, hepatocytes and monolayer epithelial Inhibitors,research,lifescience,medical cells.1,5,15,18 To the best of our knowledge, there Inhibitors,research,lifescience,medical have been no previously published

reports of siRNA transfection into MDA-MB- 468 cell line. In this method, cells are exposed to high voltage pulse in the presence of siRNA. The high voltage allows the foreign nucleic acid to enter the permeabilized cellular membrane. The first and important step in siRNA transfection by electroporation is to determine optimal electroporation condition for genetic modification, because the condition of electroporation for each cell is different. The second step in siRNA transfection is finding an optimal concentration of siRNA. Usually, the best concentration Bay 11-7085 should be determined experimentally. In this study, high transfection efficiency for the MDA-MB-468 breast cancer cell line was described. It was achieved firstly by identifying the most favorable electroporation waveform (square or exponential decay) and then by refining other parameters such as voltage, capacity and pulse duration. The viability of the cells was monitored once after electroporation by trypan blue staining and then 24 h after electroporation by MTT assay.