Aktive edilmiş dendrimerler ve G418 ayrımı ile yapılan kalıcı transfeksiyonla yeşil flöresan protein ekspresse eden CHO hücrelerinin yapılması

Background. Stable transfection of Green Fluorescent Protein (GFP) has been widely used as a marker for both monitoring protein expression and direct observations of cellular dynamics. Despite the proven utility of GFP as a marker, the appropriate conditions of stable transfection, like selection of optimal concentration of G418 and the effect of passage number on fluorescence emission time has not been fully described for stable transfection of kanamycin/neomycin resistant pIRES-EGFP to Chinese Hamster Ovary (CHO) cells by activated dendrimers. Methods. To address the issues and the establishment of GFP expressing CHO cells for further experiments, we transfected the cells with pIRES2-EGFP by using activated dendrimers. Transfected cells were selected by eluding the reminder population by exposing to G418 (500 μg/ml for 3 weeks). Selected transfected cell strains were imaged by using confocal microscopy. Results. After 48 hours following transfection, 5% of cells were transiently transfected and the emitted green fluorescence faded within 4 days. Following G418 treatment for 3 weeks, 10% of the survived cells expressed GFP with a fluorescence intensity several folds larger than that in the control cells. GFP expressing cells had complex morphology which is different from the typical fusiform shape of CHO cells. A part of stably transfected cells showed some important changes in their morphology even though no green fluorescence was detected. Conclusions. This study demonstrates that fluorescent proteins can succesfully be expressed in mammalian cells for multiple purposes. Our future goal is to obtain stable GFP expression in different cell lines to be used for different experimental purposes.