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 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.


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.
Novaković
S
S
Department of Tumor Biology, Institute of Oncology, Zaloska 2, 1000 Ljubljana, Slovenia. snovakovic@onko-i.si
Cegovnik
U
U
Menart
V
V
Galvani
V
V
Wraber
B
B
D000818:Animals; D019496:Cancer Vaccines / Q000627:therapeutic use; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008546:Melanoma, Experimental / Q000378:metabolism / Q000628:therapy; D051379:Mice; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D014162:Transfection / Q000379:methods; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics*
D019496:Cancer Vaccines; D014409:Tumor Necrosis Factor-alpha