Author/Editor		Čegovnik, Urška
Title		Priprava ekspresijske kasete z genom hTNFalfa za prehodno izražanje v sesalskih celicah
Type		monografija
Place		Ljubljana
Publisher		Univerza v Ljubljani, Medicinska fakulteta
Publication year		2000
Volume		str. 99
Language		slo
Abstract		Considering that cancer is essentially a genetic disease, different gene therapy strategies for cancer treatment have rapidly expanded in the past few years. Preliminary results are promising although it is likely that the methods for gene transfer will need further elaboration. Our aim was to create genetically modified syngeneic tumor cells capable of hTNFalpha production and excretion. The hTNFalpha gene was selected due to the complex antitumor action of the hTNFalpha protein that has at least a direct cytotoxic effect upon tumor cells, as well as indirect activity via stimulation of antitumor immunity. The first step in the creation of genetically modified tumor cells was a successful insertion of gene coding for hTNFalpha into the expression vector (creation of the expression cassette). The peDNA3 was selected as expression vector because of its high level and stable transient expression in eucaryotic cells. The vector pcDNA3 was amplified in E.coli and linearized with the restriction enzymes HindIII and EcoRI. Human TNFalpha gene was amplified by PCR, using the primers that contained restriction sites for the above-mentioned restriction enzymes. In addition, the amplified hTNFalpha gene was ligated into the vector pcDNA3 with the overlapping ends. The constructed expression vector was transferred by chemical transformation into the E. coli cells where replication occurred. The plasmid was then isolated and analyzed by PCR, restriction enzymes and sequence determination. The PCR and restriction analyses, confirmed the presence of hTNFalpha gene in the plasmid, while the sequencing confirmed the real orientation of the gene and order of its nucleotides. The expression cassette was then transferred by receptor mediated gene transfer into the mouse melanoma B 16 cells; the cell growth medium was daily collected and tested for the presence of excreted hTNFalpha protein. (Abstract truncated at 2000 characters).
Descriptors		NEOPLASMS GENE THERAPY TUMOR NECROSIS FACTOR CANCER VACCINES TUMOR CELLS, CULTURED GENE EXPRESSION REGULATION, NEOPLASTIC ESCHERICHIA COLI EUKARYOTIC CELLS GENE AMPLIFICATION POLYMERASE CHAIN REACTION MELANOMA, EXPERIMENTAL ENZYME-LINKED IMMUNOSORBENT ASSAY TRANSFECTION GENE TRANSFER