Author/Editor		Čegovnik, Urška
Title		Priprava ekspresijskih kaset za prehodno tvorbo citokinov in rastnih faktorjev v sesalskih celicah
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2003
Volume		str. 145
Language		slo
Abstract		One of the most recent and promising immunotherapeutic strategies for the treatment of cancer is based on the creation of genetically manipulated tumor vaccine. In attempts to stimulate the immune sistem in the process of its antitumor response, tumor cells were genetically modified with genes for cytokines and growth factors such as hIL2, hIFNgamma, hTNFalpha, hGMCSF and hGCSF. Antitumor activity of the mentioned cytokines and growth factors was demonstrated by the activation of specific immune system and an increase of immunogenicity of tumor cells as well as by direct cytotoxic effect of hTNFalpha on tumor and endothelial cells. The antitumor activity of cytokines was also enhanced by its synergistic effect. For that purpose, we created genetically modified tumor cells capable of two citokines/growth factors excretion. We applied two different types of expression casettes that were transfered into the same tumor cells and obtained genetically modified tumor cells with the following combination of cytokines: IL2/TNFalpha, IL2/GMCSF and TNFalpha/IFNgamma. The first step in the creation of genetically modified tumor cells was the construction of expression casettes. We inserted selected genes for cytokines/growth factors into suitable expression vectors, viz. pcDNA3, pcDNA3.1 and pVP22, which are known to have high level and stable transient expression in tumor cells. In contrast with pcDNA3 and pcDNA3.l, the pVP22 vector contained the gene for herpes simplex virus-1 structural protein named VP22. The fusion of the cloned gene with the VP22 gene permits the translocation of fused protein into the nucleus of unmodified cells in a culture that enhanced the therapeutic effect of cytokines on neighbouring cells. The genes for cytokines and growth factors were amplified by PCR. The coding region of PCR product was determined by specially constructed PCR primers. (Abstract truncated at 2000 characters).
Descriptors		CELL LINE ESCHERICHIA COLI PLASMIDS TUMOR CELLS, CULTURED CYTOKINES GROWTH SUBSTANCES TRANSFECTION GENE EXPRESSION REGULATION, NEOPLASTIC POLYMERASE CHAIN REACTION