Gene Electrotransfer of Canine Interleukin 12 into Canine Melanoma Cell Lines
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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.
KeywordsCanine IL-12 Canine melanoma cell lines Electroporation Gene electrotransfer Kanamycin Plasmid DNA
The authors would like to acknowledge M. Lavric for her help with cell cultures. This work was financially supported by the Slovenian Research Agency (programs P3-0003 and P1-0140, Projects J3-4259, J3-6796 and J3-6793). The research was conducted within the scope of LEA EBAM (French-Slovenian European Associated Laboratory: Pulsed Electric Fields Applications in Biology and Medicine) and within the COST TD1104 Action.
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