Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells

Background: Tumor vaccines, which are created by the insertion of cDNA enco ding different cytokines into the tumor cells, aree capable of inducing a v ery complex immune reaction including activation of CD8 T cells, granulocyt es, macrophages, the triggering of cytokine cascades and antibody productio n. Aiming to create genetically modified tumor cells which could produce an d secrete Human Tumor Necrosis Factor-alpha (hTNF-alpha), we constructed th e expression cassette containing hTNF-alpha gene in pcDNA3 plasmid vector: Materials and Methods: Tile successful ligation of cDNA encoding for hTNF-a lpha into pcBNA3 plasmid vector was confirmed by PCR, restriction mapping a nti sequence determination. The constructed expression cassette in pcDNA3 v ector was than transferred in vitro into malignant melanoma B16 tumor cells by the method of Receptor Mediated Gene Transfer (RMGT). Results: Measurab le amounts of hTNF-alpha protein detected in the medium of transfected cell s proved that tumor cells modified in this manner became producers of hTNF- alpha protein. Conclusion: The expression of the transferred gene was trans ient and the produced protein was biologically active. Furthermore, the pro duction of hTNF-alpha protein was also observed in sub-lethally irradiated tumor cells, showing that the expression cassette was preserved during the irradiation and that the cells were potentially applicable as a tumor vacci ne.