در القاء مقاومت و حساسيت به داروهاي زيوسين و گنسيكلووير در سلول هاي حيواني PCCI بررسي توانايي پلاسميد

Evaluation of pCCI Plasmid in Induction of Resistance and Susceptibility to Zeocin and GCV in Mammalian Cells

Plasmids and viruses are vectors capable of inducing expression of different genes in different cells. Marker genes are usually included in vectors construction in order to allow the selection of target cells from other cells. HSV.Tk-Zeo is a fusion gene that simultaneously encodes two HSV.TK and Zeo selection markers conferring susceptibility to GCV and resistance to zeocin, respectively. Vector pCCI, is a retroviral based plasmid encoding HSV.tk-Zeo fusagene that, due to containing a multiple cloning site is capable of inducing and expressing other genes of interest in host cells. Therefore, this plasmid can be used for the expression of different genes and proteins along with the HSV.TK-Zeo protein. In this article we have described the ability of pCCI plasmid in the expression of recombinant HSV.TK-Zeo fusion protein and induction of zeocin resistance and GCV susceptibility in mammalian cells. In the present study HeLa and PA-1 cells were transfected with pCCI plasmid and then the transfected cells were proliferated. Resistance of transfected cells to zeocin and their sensitivity to GCV were assayed using MTT assay. In this study untransfected cells were used as negative control. Data analysis showed that pCCI could induce zeocin resistance in transfected cells and these cells showed a great resistance to zeocin compared to untransfected cells. Transfected HeLa cells were 500-fold and PA-1 cells 100-fold more susceptible to GCV cytotoxicity compared to untransfected cells. Results confirmed that the constructed plasmid is able to induce zeocin resistance and GCV susceptibility, simultaneously. In conclusion it can be said that since pCCI is able to induce chimeric HSV.TK-Zeo marker properly, this vector and the viruses made from it can be employed in HSV.TK/GCV gene therapy protocols. Inclusion of HSV.TK with Zeo marker provides a proper system for double isolating of target cells. Moreover, the presence of a multiple cloning site for the cloning of different genes provides a biological tool for combination of suicide effect of HSV.TK and other genes of therapeutic interest which have synergy with HSV.TK. Combination of V.TK and other genes increases the cytotoxic potency of vectors made from this plasmid in killing target and specially cancer cells.