Transfection of CCE embryonic stem cells with EGFP and BDNF genes by electroporation method
F. Fathi, T. Altiraihi, J. Mowla, M. Movahedin
Dept. of Anatomy, Faculty of medical sciences, Kurdistan medical university, Pasdaran Street, Sanandaj, IRAN
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.
Key words:
Embryonic Stem Cells, Transfection, Electroporation, GFP, BDNF
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