Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues.
Antibiotic resistance gene-free
Copy number
Gene electrotransfer
Interleukin 12
Plasmid
Transfection efficiency
Journal
Heliyon
ISSN: 2405-8440
Titre abrégé: Heliyon
Pays: England
ID NLM: 101672560
Informations de publication
Date de publication:
Feb 2022
Feb 2022
Historique:
received:
29
11
2021
revised:
29
12
2021
accepted:
29
01
2022
entrez:
10
3
2022
pubmed:
11
3
2022
medline:
11
3
2022
Statut:
epublish
Résumé
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, European 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
Identifiants
pubmed: 35265755
doi: 10.1016/j.heliyon.2022.e08879
pii: S2405-8440(22)00167-0
pmc: PMC8899673
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e08879Informations de copyright
© 2022 The Authors.
Déclaration de conflit d'intérêts
The authors declare no conflict of interest.
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