Evaluation of the efficiency of pIRES2 EGFP and pcDNA3-hBDNF-v5 plasmids in transfection of CCE ES cells by the electroporation method

ABSTRACT

Unlimited self renewal and potential capacity of embryonic stem cells [ESCs] 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. The aim of the present study was the evaluation of the transfection efficiency of pIRES2-EGFP and pcDNA3-hBDNF-v5 plasmids in CCE ES cells by the electroporation method. The plasmids transformed into DH5 bacteria and propagated as maxi-prep. The plasmids then purified and transfected into ESCs by means of the electroporation method. To confirm the expression of the GFP and BDNF genes, invert fluorescent microscopy and RT-PCR were used. Expression of EGFP was confirmed by examining the transfected cells with fluorescent microscopy. In case of BDNF, total RNA extracted from stably transfected cells, were quantitatively evaluated by spectrophotometry and qualitatively by agarose gel electrophoresis. Then mRNAs were reverse transcripted and BDNF cDNA amplified by specific primers. The products of the PCR were 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 the CMV promoter is active in undifferentiated mouse ESCs and a CCE cell line is an appropriate donor cell for cell-mediated BDNF gene transfer