Avtor/Urednik		Novaković, S; Čegovnik, U; Menart, V; Galvani, V; Wraber, B
Naslov		Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells
Tip		članek
Vir		Anticancer Res
Vol. in št.		Letnik 21, št. 1A
Leto izdaje		2001
Obseg		str. 365-71
Jezik		eng
Abstrakt		Background: Tumor vaccines, which are created by the insertion of cDNA encoding different cytokines into the tumor cells, are capable of inducing a very complex immune reaction including activation of CD8 T cells, granulocytes, macrophages, the triggering of cytokine cascades and antibody production. Aiming to create genetically modified tumor cells which could produce and secrete Human Tumor Necrosis Factor-alpha (hTNF-alpha), we constructed the expression cassette containing hTNF-alpha gene in pcDNA3 plasmid vector. Mathearials and methods: The successful ligation of cDNA encoding for hTNF-alpha into pcDNA3 plasmid vector was confirmed by PCR, restriction mapping and sequence determination. The constructed expression cassette in pcDNA3 vector was than transferred in vitro into malignant melanoma B16 tumor cells by the method of Receptor Mediated Gene Transfer (RMGT). Results: Measurable amounts of hTNF-alpha protein detected in the medium of transfected cells proved that tumor cells modified in this manner became producers of hTNF-alpha protein. Conclusions: The expression of the transferred gene was transient and the produced protein was biologically active. Furthermore, the production 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 vaccine.
Deskriptorji		TUMOR NECROSIS FACTOR CANCER VACCINES CLONING, MOLECULAR GENETIC VECTORS MELANOMA, EXPERIMENTAL MICE PLASMIDS RESTRICTION MAPPING TRANSFECTION TRANSGENES