The Journal of Membrane Biology

, Volume 248, Issue 5, pp 909–917 | Cite as

Gene Electrotransfer of Canine Interleukin 12 into Canine Melanoma Cell Lines

  • Ursa Lampreht
  • Urska Kamensek
  • Monika Stimac
  • Gregor Sersa
  • Natasa Tozon
  • Masa Bosnjak
  • Andreja Brozic
  • Geraldo Gileno de Sá Oliveira
  • Takayuki Nakagawa
  • Kohei Saeki
  • Maja Cemazar
Article

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

Canine IL-12 Canine melanoma cell lines Electroporation Gene electrotransfer Kanamycin Plasmid DNA 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ursa Lampreht
    • 1
  • Urska Kamensek
    • 1
  • Monika Stimac
    • 1
  • Gregor Sersa
    • 1
  • Natasa Tozon
    • 2
  • Masa Bosnjak
    • 1
  • Andreja Brozic
    • 3
  • Geraldo Gileno de Sá Oliveira
    • 4
  • Takayuki Nakagawa
    • 5
  • Kohei Saeki
    • 5
  • Maja Cemazar
    • 1
    • 6
  1. 1.Department of Experimental OncologyInstitute of Oncology LjubljanaLjubljanaSlovenia
  2. 2.Small Animal Clinic, Veterinary FacultyUniversity of LjubljanaLjubljanaSlovenia
  3. 3.Department of CytopathologyInstitute of Oncology LjubljanaLjubljanaSlovenia
  4. 4.Laboratory of Pathology and Bio-Intervention, Goncalo Moniz Research Center, Oswaldo Cruz FoundationNational Institute of Science and Technology of Tropical Diseases (INCT-DT)SalvadorBrazil
  5. 5.Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life sciencesThe University of TokyoTokyoJapan
  6. 6.Faculty of Health SciencesUniversity of PrimorskaIzolaSlovenia

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