Author/Editor     Lampreht Tratar, Urša; Kamenšek, Urška; Štimac, Monika; Serša, Gregor; Tozon, Nataša; Bošnjak, Maša; Brožič, Andreja; Oliveira, Geraldo Gileno de Sá; Nakagawa, Takayuki; Saeki, Kohei; Čemažar, Maja
Title     Gene electrotransfer of canine interleukin 12 into canine melanoma cell lines
Type     članek
Vol. and No.     Letnik 248, št. 5
Publication year     2015
Volume     str. 909-917
ISSN     1432-1424 - The Journal of membrane biology
Language     eng
Abstract     A gene electrotransfer (GET) of interleukin 12 (IL-12) had already given good results when treating tumors in human and veterinary clinical trials. So far, plasmids used in veterinary clinical studies encoded a human or a feline IL-12 and an ampicillin resistance gene, which is not recommended by the regulatory agencies to be used in clinical trials. Therefore, the aim of the current study was to construct the plasmid encoding a canine IL-12 with kanamycin antibiotic resistance gene that could be used in veterinary clinical oncology. The validation of the newly constructed plasmid was carried out on canine malignant melanoma cells, which have not been used in GET studies so far, and on human malignant melanoma cells. Canine and human malignant melanoma cell lines were transfected with plasmid encoding enhanced green fluorescence protein at different pulse parameter conditions to determine the transfection efficiency and cell survival. The IL-12 expression of the most suitable conditions for GET of the plasmid encoding canine IL-12 was determined at mRNA level by the qRT-PCR and at protein level with the ELISpot assay. The obtained results showed that the newly constructed plasmid encoding canine IL-12 had similar or even higher expression capacity than the plasmid encoding human IL-12. Therefore, it represents a promising therapeutic plasmid for further IL-12 gene therapy in clinical studies for spontaneous canine tumors. Additionally, it also meets the main regulatory agencies% (FDA and EMA) criteria.
Keywords     interlevkin-12
melanom
psi
elektroporacija
elektrogenska terapija
canine IL-12
canine melanoma cell lines
electroporation
gene electrotransfer