Transfection of CCE mouse embryonic stem cells with EGFP and BDNF genes by the electroporation method.
Embryonic stem (ES) cells are pluripotent cells that can differentiate into a wide variety of cell types. This has made them 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 genetic manipulation of the cells. The aim of the present study was to transfect CCE ES cells with EGFP and BDNF genes, in which pcDNA3-hBDNF-v5 and pIRES2-EGFP plasmids were used, respectively. Transformation of DH5alpha competent bacteria by the plasmids was done. Then the plasmids were purified and transfected into ES cells using the electroporation method. The expression of the EGFP gene was confirmed using invert fluorescent microscopy; accordingly, RT-PCR was used for the BDNF gene. The latter was evaluated by extracting the total RNA from the transfected cells. cDNA was obtained by using reverse transcriptase and was amplified by specific primers. The products of the PCR were separated and visualized by 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.
Fathi
Fardin
F
Department of Anatomical Sciences, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran.
Tiraihi
Taki
T
Mowla
Seyed Javad
SJ
Movahedin
Mansoureh
M
D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics*; D018274:Electroporation; D004622:Embryo, Mammalian / Q000166:cytology; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection / Q000379:methods*
D019208:Brain-Derived Neurotrophic Factor; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins