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  • Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues [Elektronski vir]
    Kamenšek, Urška ...
    Interleukin 12 (IL-12) is a cytokine used as a therapeutic molecule in cancer immunotherapy. Gene electrotransfer mediated delivery of IL-12 gene has reached clinical evaluation in the USA using a ... plasmid that in addition to IL- 12 gene also carry an antibiotic resistance gene needed for its production in bacteria. In Europe however, Eu- ropean Medicines Agency recommends against the use of antibiotics during the production of clinical grade plasmids. We have prepared several antibiotic resistance gene-free plasmids using an antibiotic-free selection strategy called operator-repressor titration, including plasmids encoding mouse, canine and human IL-12 orthologues. The aim of this study was to evaluate the maintenance of these plasmids in bacterial culture and test their transfection efficiency using gene electrotransfer. Plasmid maintenance was evaluated by determining plasmid yields and topologies after subculturing transformed bacteria. Transfection efficiency was evaluated by determining the plasmid copy number, expression and cytotoxicity after gene electrotransfer to mouse, canine and human melanoma cells. The results demonstrated that our IL-12 plasmids without an antibiotic resistance gene are stably maintained in bacteria and provide sufficient IL-12 expression after in vitro gene electrotransfer; therefore, they have the potential to proceed to further in vivo evaluation studies.
    Source: Heliyon [Elektronski vir]. - ISSN 2405-8440 (Vol. 8, no. 2, Feb. 2022, str. 1-08879-10-08879)
    Type of material - e-article
    Publish date - 2022
    Language - english
    COBISS.SI-ID - 96531715

    Link(s):

    https://doi.org/10.1016/j.heliyon.2022.e08879
    DiRROS - Digital repository of Slovenian research organizations
    DOI

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