Unlimited self renewal and potential capacity of embryonic stem cells (ESC) in differentiating into a wide variety of cell types has made the cells an attractive source of donor cells for developmental studies and cell therapy. Blocking the differentiation of ES cells in culture and using them in clinical applications requires some genetic manipulations of the cells. The aim of the present study is to transfect CCE ES cells by EGFP and BDNF Genes. For this purpose pIRES2-EGFP and pcDNA3-hBDNF-v5 plasmids were used. At first the plasmids transformed into DH5α competent bacteria and propagated as maxi-prep. The plasmid then purified and transfected into ES cells by means of electroporation method. To confirm the expression of the EGFP and BDNF genes, invert fluorescent microscopy and RT-PCR were used, respectively. Expression of EGFP confirmed by examining the transfected cells with flourscent microscopy. In case of BDNF, total RNA extracted from stably transfected cells, evaluated quantitatively by spectrophotometry and qualitatively by agarose gel electrophoresis. Then mRNAs reverse transcriptized and BDNF cDNA amplified by specific primers. The products of the PCR separated and visualized on agarose gel electrophoresis. Both techniques revealed a successful transfection of CCE ES cells by both plasmids. The obtained data indicated that electroporation is an efficient method for transfection of CCE ES cells. Our data also show that the CCE cell line is an appropriate donor cell for cell-mediated gene transfer.but pires2-EGFP plasmid isnt a good plasmid for stable transfection of ES Cells.
Embryonic Stem Cells